Method of Treating or Preventing Tissue Deterioration, Injury or Damage Due to Disease of Mucosa

ABSTRACT

An immunomodulatory compound is utilized to treat mucosa disease.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application No. 60/900,977, filed Feb. 13, 2007.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of mucosa disease treatment.

2. Description of the Background Art

Oral ulcerative mucositis is a common, painful, dose-limiting toxicity of drug and radiation therapy for cancer. The disorder is characterized by breakdown of the oral mucosa that results in the formation of ulcerative lesions. In granulocytopenic patients, the ulcerations that accompany mucositis are frequent portals of entry for indigenous oral bacteria often leading to sepsis or bacteremia. Mucositis occurs to some degree in more than one third of patients receiving anti-neoplastic drug therapy. The frequency and severity are significantly greater among patients who are treated with induction therapy for leukemia or with many of the conditioning regimens for bone marrow transplant. Among these individuals, moderate to severe mucositis is not unusual in more than three-quarters of patients. Moderate to severe mucositis occurs in virtually all patients who receive radiation therapy for tumors of the head and neck and typically begins with cumulative exposures of 15 Gy and then worsens as total doses of 60 Gy or more are reached.

Clinically mucositis progresses through three stages:

1. Inflammation accompanied by painful mucosal erythema, which can respond to local anesthetics. 2. Painful ulceration with pseudomembrane formation and, in the case of myelosuppressive treatment, potentially life-threatening sepsis, requiring antimicrobial therapy. Pain is often of such intensity as to require parenteral narcotic analgesia. 3. Spontaneous healing, occurring about 2-3 weeks after cessation of anti-neoplastic therapy.

Standard therapy for mucositis is predominantly palliative, including application of topical analgesics such as lidocaine and/or systemic administration of narcotics and antibiotics. Currently, there is only one approved treatment for oral mucositis, Kepivance (Palifermin), which is only approved for the treatment of oral mucositis in patients undergoing conditioning regimens prior to hematopoetic stem cell transplantation for the treatment of hematologic malignancies.

The complexity of mucositis as a biological process has only been recently appreciated. It has been suggested that the condition represents a sequential interaction of oral mucosal cells and tissues, reactive oxygen species, pro-inflammatory cytokines, mediators of apoptosis and local factors such as saliva and the oral micro biota. While epithelial degeneration and breakdown ultimately result in mucosal ulceration, it appears that the early changes associated with radiation-induced mucosal toxicity occur within the endothelium, and connective tissue of the submucosa. Electron microscopic evaluation of mucosa within 1 week of radiation shows damage to both endothelium and connective tissue, but not epithelium. Such injury is likely mediated by free radical formation. It appears that the overall mechanism for mucositis development is similar for both radiation and chemotherapy.

There remains a need in the art for improvements in methods for treating or preventing mucosa diseases.

SUMMARY OF THE INVENTION

In accordance with one aspect, a method of treatment for treating, preventing, inhibiting or reducing tissue deterioration, injury or damage due to disease of mucosa, or for restoring tissue adversely affected by said disease, in a subject, comprises administering to said target subject an effective amount of an immunomodulator compound of formula A:

In Formula A, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptide fragment, and X is an aromatic or heterocyclic amino acid or a derivative thereof, wherein said immunomodulator compound is administered to said target subject without administering said at least one interferon to said target subject. Preferably, X is L-tryptophan or D-tryptophan. The invention also relates to use of a compound of formula A in preparation of a medicament for treatment of mucosa disease.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is applicable to mucosa diseases or disease manifestations thereof. In preferred embodiments, the mucosa disease results from radiation and/or chemotherapy of a subject, preferably in a human patient. The radiation may be acute or fractionated. The mucosa disease may result from a combination of radiation and chemotherapy. In certain embodiments, the disease is of oral and/or esophageal mucosa, e.g., mucositis and/or ulcerative lesions.

In accordance with one embodiment, the present invention relates to treatment of mucositis by administering an immunomodulator compound to a mammalian subject, preferably a human patient.

In certain embodiments, the immunomodulatory compounds of the present invention are administered before, during and/or after administration of radiation and/or a chemotherapeutic agent to a patient. Radiation often is administered in multiple doses, and mucosa disease occurs in virtually all patients who receive radiation therapy for tumors of the head and neck, typically beginning with cumulative exposures of 15 Gy radiation and then worsening at total cumulative doses of 60 Gy radiation or more. In preferred embodiments, an immunomodulator compound according to the invention is administered before, during or after administration of 7-8 Gy (e.g., 7.5 Gy) radiation to a patient, 15 Gy, 40 Gy, 60 Gy, or more radiation to patient.

The immunomodulator compounds of the invention also can be administered for the treatment or prevention of mucosa disease resulting from administration of chemotherapy agents, such as cis-platin, e.g., administered at a dosage within a range of about 0.1-50 mg/kg, e.g., about 5 mg/kg.

Immunomodulator compounds used in accordance with the present invention, comprise immunomodulators of Formula A:

In Formula A, n is 1 or 2, R is hydrogen, acyl, alkyl or a peptide fragment, and X is an aromatic or heterocyclic amino acid or a derivative thereof. Preferably, X is L-tryptophan or D-tryptophan. Appropriate derivatives of the aromatic or heterocyclic amino acids for “X” are: amides, mono-or di-(C₁-C₆) alklyl substituted amides, arylamides, and (C₁-C₆) alkyl or aryl esters. Appropriate acyl or alkyl moieties for “R” are: branched or unbranched alkyl groups of 1 to about 6 carbons, acyl groups from 2 to about 10 carbon atoms, and blocking groups such as carbobenzyloxy and t-butyloxycarbonyl. Preferably the carbon of the CH group shown in Formula A has a stereoconfiguration, when n is 2, that is different from the stereoconfiguration of X.

Preferred embodiments utilize compounds such as γ-D-glutamyl-L-tryptophan, γ-L-glutamyl-L-tryptophan, γ-L-glutamyl-N_(in)-formyl-L-tryptophan, N-methyl-γ-L-glutamyl-L-tryptophan, N-acetyl-γ-L-glutamyl-L-tryptophan, γ-L-glutamyl-D-tryptophan, β-L-aspartyl-L-tryptophan, and β-D-aspartyl-L-tryptophan. Particularly preferred embodiments utilize γ-D-glutamyl-L-tryptophan, sometimes referred to as SCV-07. These compounds, methods for preparing these compounds, pharmaceutically acceptable salts of these compounds and pharmaceutical formulations thereof are disclosed in U.S. Pat. No. 5,916,878, incorporated herein by reference.

SCV-07, γ-D-glutamyl-L-tryptophan, is a member of a class of immunomodulatory drugs that possess γ-glutamyl or β-aspartyl moieties, which was discovered by Russian scientists and is being examined for efficacy in several indications in the U.S. by SciClone Pharmaceuticals, Inc. SCV-07 possesses a number of immunomodulatory activities in vivo and in vitro. SCV-07 increases Con-A-induced thymocyte and lymphocyte proliferation, increases Con-A-induced interleukin-2 (IL-2) production and IL-2 receptor expression by spleen lymphocytes, and stimulates expression of Thy-1.2 on bone marrow cells. In vivo, SCV-07 has a strong immunostimulatory effect on 5-FU-immune-suppressed animals and in a model of immunization with sheep red blood cells.

The Formula A compounds may be administered as dosages in the range of about 0.001-2000 mg, more preferably about 0.01-100 mg. Dosages may be administered one or more times per week, preferably on a daily basis, with dosages administered one or more times per day. Administration can be by any suitable method, including orally, nasally, transdermally, sublingually, by injection, periodic infusion, continuous infusion, and the like. The dosages may be administered by intramuscular injection, although other forms of injection and infusion may be utilized, and other forms of administration such as oral or nasal inhalation or oral ingestion may be employed.

Dosages may also be measured in micrograms per kilogram, with dosages in the range of about 0.1-10,000 ug/kg, more preferably within the range of about 1.0-1000 ug/kg.

Included are biologically active analogs having substituted, deleted, elongated, replaced, or otherwise modified portions which possess bioactivity substantially similar to that of SCV-07, e.g., an SCV-07 derived peptide having sufficient homology with SCV-07 such that it functions in substantially the same way with substantially the same activity as SCV-07.

In some embodiments, the Formula A compound is present in a pharmaceutically acceptable liquid carrier, such as water for injection, saline in physiological concentrations, or similar, or in tablet form with suitable dry carrier(s) and excipient(s).

Effective amounts of Formula A compound can be determined by routine dose-titration experiments.

Example 1 A First Study of SCV-07 in the Treatment of Oral Mucositis Induced by Acute Radiation in Hamsters 1. Objective

The objective of this study was to conduct a preliminary evaluation of the efficacy of SCV-07 in the treatment of oral mucositis using an acute radiation induced hamster model of the disease. SCV-07 was given at doses of 1, 10 or 100 ug/kg once daily by sub-cutaneous injection for 18 days, starting one day before radiation and continuing until day 16 after radiation. No deaths were observed in any of the treatment groups and there were no statistically significant changes in growth rate, suggesting that SCV-07 was well tolerated at these doses. Animals treated with SCV-07 at 10 ug/kg on days −1 to 16 showed a statistically significant reduction in mucositis scores on day 22 (P=0.024). Animals treated with SCV-07 at 100 ug/kg on days −1 to 16 showed a statistically significant reduction in mucositis scores on day 14 (P=0.025), in addition to a statistically significant reduction in the number of animal days with a score of 3 or higher (P=0.029). These data suggest that SCV-07 demonstrated a dose dependant benefit on the severity and course of radiation induced mucositis.

Acute Radiation Model

The acute radiation model in hamsters has proven to be an accurate, efficient and cost-effective technique to provide a preliminary evaluation of anti-mucositis compounds. The course of mucositis in this model is well defined and results in peak scores approximately 14-16 Days following radiation. The acute model has little systemic toxicity, resulting in few hamster deaths, thus permitting the use of smaller groups (N=7-8) for initial efficacy studies. It has also been used to study specific mechanistic elements in the pathogenesis of mucositis. Molecules that show efficacy in the acute radiation model may be further evaluated in the more complex models of fractionated radiation, chemotherapy, or concomitant therapy.

In this study, an acute radiation dose of 40 Gy on day 0 was administered. Clinically significant mucositis was observed on days 12 through 28.

2. Study Objective and Summary Study Objective

The objective of this study was to evaluate the effect of SCV-07, administered by sub-cutaneous injection on different schedules between the day before radiation and the sixteenth day after radiation, on the frequency, severity and duration of oral mucositis induced by acute radiation.

Study Summary

Thirty-two (32) Syrian Golden Hamsters were given an acute radiation dose of 40 Gy directed to their left buccal cheek pouch on day 0. Test materials were given by sub-cutaneous injection once daily. Dosing began one day before radiation (day −1) and continued until day 16. Mucositis was evaluated clinically starting on day 6, and continuing on alternate days until day 28.

3. Evaluation Mucositis Evaluation

The grade of mucositis was scored, beginning day 6, and for every second day thereafter, through and including day 28. The effect on mucositis of each drug treatment compared to placebo was assessed according to the following parameters:

The difference in the number of days hamsters in each group have ulcerative (score ≧3) mucositis.

On each evaluation day, the number of animals with a blinded mucositis score of ≧3 in each drug treatment group was compared to the control group. Differences were compared on a cumulative basis and statistical significance was determined by chi-square analysis. Efficacy, in this analysis, is defined by a significant reduction in the number of days that a group of animals had ulcerations (scores ≧3) when compared to the control group.

Rank Sum Differences in Daily Mucositis Scores

For each evaluation day the scores of the control group were compared to those of the treated groups using non-parametric rank sum analysis. Treatment success was considered as a statistically significant lowering of scores in the treated group on 2 or more days from day 6 to day 28.

Weights and Survival

All animals were weighed daily and their survival recorded, in order to assess possible differences in animal weight among treatment groups as an indication for mucositis severity and/or possible toxicity resulting from the treatments.

4. Study Design

Thirty-two Syrian Golden Hamsters were divided into four (4) groups of eight (8) animals each. All animals received a single dose of acute radiation of 40 Gy directed to their left buccal cheek pouch on day 0. This was accomplished by anesthetizing the animals and everting the left buccal pouch, while protecting the rest of the animals with a lead shield. Test materials were given by sub-cutaneous injection once daily as detailed in Table 1. Mucositis was evaluated clinically starting on Day 6, and continuing on alternate days until day 28.

TABLE 1 Study Design Group Number of Treatment Volume Number Animals Treatment Schedule* (mL) 1 8 males Vehicle (PBS), sc, Day −1 to 16 0.1 mL/100 g qd 2 8 males SCV-07, sc, qd Day −1 to 16 0.1 mL/100 g 1 μg/kg, 3 8 males SCV-07, sc, qd Day −1 to 16 0.1 mL/100 g 10 μg/kg 4 8 males SCV-07, sc, qd Day −1 to 16 0.1 mL/100 g 100 μg/kg *The dose on day 0 will be performed 30 minutes prior to radiation

5. Material and Methods Location of Study Performance

The study was performed at Biomodels AAALAC accredited facility in Cambridge Mass. Approval for this study was obtained from Biomodels IACUC.

Animals

Male LVG Syrian Golden Hamsters (Charles River Laboratories), aged 5 to 6 weeks, with average body weight of 85.3 g at study commencement, were used. Animals were individually numbered using an ear punch and housed in small groups of 8 animals per cage. Animals were acclimatized prior to study commencement. During this period of 5 days, the animals were observed daily in order to reject animals that present in poor condition.

Housing

The study was performed in animal rooms provided with filtered air at a temperature of 70° F.+/−5° F. and 50%+/−20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. Bed-O-Cobs® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity was recorded during the study, and the records retained.

Diet

Animals were fed with a Purina Labdiet® 5061 rodent diet and water was provided ad libitum.

Animal Randomization and Allocations

Animals were randomly and prospectively divided into four (4) treatment groups prior to irradiation. Each animal was identified by an ear punch corresponding to an individual number. For more consistent identification, ear punch numbering was used rather than tagging, since tags may become dislodged during the course of the study. A cage card was used to identify each cage or label marked with the study number (SCI-01), treatment group number and animal numbers.

Sub-Cutaneous Dosing and Drug Application

The test compound, SCV-07 was provided as a powder and dissolved in sterile PBS immediately prior to administration. Three dilutions were prepared: 100 μg/mL, 10 μg/mL and 1 μg/mL. Drug was given in a volume of 0.1 mL per 100 g body weight, with the appropriate dilution of SCV-07 for each group, using a tuberculin syringe with a 27G needle. Injections were given subcutaneously to the back or abdomen.

Mucositis Induction

Mucositis was induced using a standardized acute radiation protocol. A single dose of radiation (40 Gy/dose) was administered to all animals on day 0. Radiation was generated with a 160 kilovolt potential (15-ma) source at a focal distance of 30 cm, hardened with a 0.35 mm Cu filtration system. Irradiation targeted the left buccal pouch mucosa at a rate of 3.2 Gy/minute. Prior to irradiation, animals were anesthetized with an intraperitoneal injection of Ketamine (160 mg/kg) and Xylazine (8 mg/kg). The left buccal pouch was everted, fixed and isolated using a lead shield to protect all other parts of the hamster's body.

Mucositis Scoring

The mucositis score, weight change and survival were measured throughout the study as described above. For the evaluation of mucositis, the animals were anesthetized with an inhalation anesthetic, and the left pouch everted. Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 for normal, to 5 for severe ulceration (clinical scoring). In descriptive terms, this scale is defined as follows:

TABLE 2 Mucositis Scoring. Score: Description: 0 Pouch completely healthy. No erythema or vasodilation. 1 Light to severe erythema and vasodilation. No erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of superficial aspects of mucosa leaving denuded areas. Decreased stippling of mucosa. 3 Formation of off-white ulcers in one or more places. Ulcers may have a yellow/gray due to pseudomembrane. Cumulative size of ulcers should equal about ¼ of the pouch. Severe erythema and vasodilation. 4 Cumulative seize of ulcers should equal about ½ of the pouch. Loss of pliability. Severe erythema and vasodilation. 5 Virtually all of pouch is ulcerated. Loss of pliability (pouch can only partially be extracted from mouth).

A score of 1-2 is considered to represent a mild stage of the disease, whereas a score of 3-5 is considered to indicate moderate to severe mucositis. Following visual scoring, a photograph was taken of each animal's mucosa using a standardized technique. At the conclusion of the experiment, all films were developed and the photographs randomly numbered. At least two independent trained observers graded the photographs in blinded fashion using the above-described scale (blinded scoring).

6. Results and Discussion 6.1 Survival

No deaths were observed during the course of this study.

6.2 Weight Change

The mean daily percent weight change data was evaluated. The saline (PBS) treated control hamsters gained an average of 70.4% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 1 ug/kg on days −1 to 16 gained an average of 68.4% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 10 ug/kg on days −1 to 16 gained an average of 70.3% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 100 ug/kg on days −1 to 16 gained an average of 82.0% of their starting weight during the study. The significance of these differences was evaluated by calculating the area-under-the-curve (AUC) for the weight gain of each animal, and then comparing the different treatment groups using a One-Way ANOVA test. The results of this analysis indicated that there were no significant differences between the different treatment groups (P=0.191).

6.3 Mucositis (Tables 3 & 4)

Mean daily mucositis scores for each group were calculated. In the saline treated control group, peak levels of mucositis were seen on day 20, when the mean score reached 3.8. The group receiving SCV-07 at 1 ug/kg from day −1 to 16 had a peak mean mucositis score of 3.8 on day 16. The group receiving SCV-07 at 10 ug/kg from day −1 to 16 had a peak mean mucositis score of 3.7 on day 16. The group receiving SCV-07 at 100 ug/kg from day −1 to 16 had a peak mean mucositis score of 3.4 on day 18. The significance of the differences observed between the different treatment groups was evaluated by calculating the number of days with a score of 3 or higher for each group and comparing these numbers using a chi-squared test. The results of this analysis are shown in Table 2. The hamsters in the saline treated control group had a score of 3 or higher on 46.9% of the animal days evaluated. In the group receiving SCV-07 at 1 ug/kg from day −1 to 16, a mucositis score of 3 or higher also was observed on 46.9% of the animals days evaluated. The hamsters in the groups treated with SCV-07 at 10 ug/kg on days −1 to 16 had a score of 3 or higher on 37.5% of animal days respectively, which was not statistically significantly different from the controls (P=0.079). The group treated with SCV-07 at 100 ug/kg on days −1 to 16 on days 1 to 16 had a score of 3 or higher on 35.4% of animal days, which was significantly different from controls (P=0.029).

A further analysis of the mucositis scores was performed using the Mann-Whitney rank sum analysis to compare the scores for each group on each day. The results of this analysis are shown in Table 4. In this analysis, 2 days of significant reduction in the mucositis score are generally required before it is regarded as meaningful reduction in mucositis. The group treated with SCV-07 at 1 ug/kg on days −1 to 16 did not show any statistically significant improvement on any day of the study, relative to the saline controls. The group treated with SCV-07 at 10 ug/kg on days −1 to 16 showed significant improvement relative to controls on day 22 (P=0.024). The group treated with SCV-07 at 100 ug/kg on days −1 to 16 showed significant improvement relative to controls on day 14 (P=0.025).

TABLE 3 Chi-square analysis of the total number of days the animals in each group spent with a score of three or more. This statistic is a measure of severity of ulceration, a clinically important outcome. Days Days Total % Days Chi Sq Group >=3 <3 Days >=3 v control P Value Control 90 102 192 46.9 — — SCV-07 90 102 192 46.9 0.0105 0.919 1 ug/kg sc Days −1 to 16 SCV-07 72 120 192 37.5 3.0860 0.079 10 ug/kg sc Days −1 to 16 SCV-07 68 124 192 35.4 4.7420 0.029 100 ug/kg sc Days −1 to 16

TABLE 4 The significance of group differences observed in daily mucositis scores was determined using the Mann-Whitney rank sum test. This nonparametric statistic is appropriate for the visual mucositis scoring scale. The p values for each calculation are shown. Significant improvements are shown underlined. Day Group Comparison 6 8 10 12 14 16 18 20 22 24 26 28 Control vs 0.555 0.775 0.069 0.531 0.234 0.145 0.557 0.373 0.230 0.558 0.554 0.733 SCV-07 1 ug/kg Days −1 to 16 Control vs 0.985 0.775 0.372 0.554 0.117 0.265 0.374 0.116 0.024 0.316 0.608 0.361 SCV-07 10 ug/kg Days −1 to 16 Control vs 0.555 0.985 0.776 0.261 0.025 0.061 0.112 0.100 0.461 0.192 0.484 0.531 SCV-07 100 ug/kg Days −1 to 16

Conclusions

1. There was no evidence of any toxicity from SCV-07 in this study based on the observations of mortality and weight gain. 2. Animals treated with SCV-07 at 10 ug/kg on days −1 to 16 showed a statistically significant reduction in mucositis scores on day 22 (P=0.024) that was dose related. 3. Animals treated with SCV-07 at 100 ug/kg on days −1 to 16 showed a statistically significant reduction in mucositis scores on day 14 (P=0.025), in addition to a statistically significant reduction in the number of animal days with a score of 3 or higher (P=0.029). 4. A favorable dose-dependent effect of SCV-07 on the severity and course of mucositis was seen. Increasing the dosing frequency or doubling the single daily dose might enhance the effects noted.

APPENDICES

APPENDIX 1 Animal Weights DAY Group Animal −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 77 80 78 83 85 86 91 92 96 96 98 97 102 102 101 102 1 2 87 90 92 97 99 101 106 110 113 119 118 119 124 125 125 127 1 3 89 92 91 96 99 102 107 108 113 115 111 118 126 126 122 123 1 4 81 82 82 86 87 89 94 96 96 99 102 101 107 107 106 106 1 5 83 86 86 90 89 91 94 101 99 101 106 102 108 108 108 109 1 6 82 85 87 92 94 96 99 104 106 106 112 108 110 110 111 110 1 7 82 93 93 98 102 104 109 113 115 117 120 119 124 127 126 125 1 8 88 80 79 83 84 86 92 92 97 98 100 100 103 105 104 105 2 9 78 96 98 100 101 103 109 111 114 116 118 117 122 123 122 123 2 10 94 86 86 89 91 94 97 99 99 102 104 104 109 110 110 110 2 11 93 80 79 84 88 90 91 95 96 101 101 100 104 105 104 107 2 12 79 83 83 86 86 89 94 98 100 103 104 102 105 107 109 111 2 13 82 86 88 91 93 95 100 105 103 105 107 106 111 114 112 116 2 14 84 86 88 91 93 96 100 101 104 105 107 106 111 114 111 112 2 15 79 80 81 84 84 86 91 94 94 97 99 98 105 110 108 110 2 16 79 81 78 83 84 87 89 93 94 98 100 97 102 105 104 106 3 17 81 83 84 87 89 92 96 98 100 102 104 103 111 112 111 111 3 18 87 90 91 94 98 100 105 107 108 111 112 113 117 120 118 115 3 19 85 87 87 90 92 93 97 98 101 103 105 104 109 109 109 103 3 20 76 76 76 79 82 84 89 90 92 96 96 96 101 101 100 102 3 21 90 93 93 96 98 100 103 106 108 109 111 110 113 117 113 110 3 22 91 95 94 98 100 103 108 109 112 114 116 115 121 124 122 122 3 23 86 91 91 95 98 100 105 101 109 113 114 115 121 121 121 121 3 24 91 95 96 100 104 106 113 116 115 120 122 124 129 130 130 129 4 25 84 87 87 91 93 96 100 103 105 109 111 110 117 119 117 117 4 26 87 91 98 94 95 98 101 104 105 109 112 111 119 117 115 118 4 27 97 101 101 108 110 111 119 123 124 127 132 133 137 138 134 139 4 28 88 95 93 98 101 103 101 115 118 118 124 121 130 131 132 139 4 29 86 90 86 92 97 100 104 106 108 111 114 115 120 123 121 125 4 30 84 88 89 92 95 99 106 109 111 115 120 118 129 131 127 132 4 31 95 100 99 106 109 111 118 119 122 126 127 128 136 137 133 138 4 32 85 87 89 94 94 97 105 106 108 110 114 113 118 121 118 120 DAY Group Animal 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1 1 101 103 100 107 109 109 112 113 116 115 119 119 121 123 1 2 126 125 130 134 140 141 143 146 149 149 153 152 154 158 1 3 122 129 123 130 134 135 137 140 142 147 147 148 149 152 1 4 106 109 106 115 116 117 119 121 126 125 130 127 131 136 1 5 108 110 109 118 119 121 122 124 129 128 131 132 135 137 1 6 111 113 113 120 121 121 124 127 128 130 135 135 138 140 1 7 127 128 127 137 139 141 143 144 149 149 153 153 156 159 1 8 105 108 106 114 117 117 119 123 126 126 129 129 132 135 2 9 123 129 126 133 136 137 139 141 143 143 147 148 150 152 2 10 110 115 114 118 121 120 122 125 128 129 131 134 135 138 2 11 111 112 108 117 118 118 121 123 124 124 129 131 134 135 2 12 111 116 112 119 123 122 123 124 130 131 137 133 139 138 2 13 117 121 117 125 127 128 131 132 135 133 140 140 144 147 2 14 112 115 113 119 120 122 123 125 126 128 131 131 133 136 2 15 109 113 108 116 118 119 120 123 125 126 130 130 133 137 2 16 106 111 109 115 118 120 121 121 126 125 129 131 132 135 3 17 112 116 116 122 124 123 122 123 126 126 132 132 133 137 3 18 110 102 107 113 115 117 119 121 122 126 128 130 133 137 3 19 101 111 102 107 111 112 113 115 117 120 122 123 120 129 3 20 105 107 104 113 116 115 119 119 122 122 127 127 131 134 3 21 111 115 111 117 119 120 120 123 125 125 128 129 132 135 3 22 120 124 123 130 132 134 136 137 140 139 145 145 149 151 3 23 121 124 125 132 137 139 139 141 145 146 146 153 155 137 3 24 131 134 133 142 146 147 149 150 155 155 160 161 165 167 4 25 116 118 118 124 124 124 123 124 124 124 127 127 129 131 4 26 117 120 119 126 126 129 132 134 135 135 138 140 143 147 4 27 138 141 142 151 153 154 156 157 161 167 167 175 169 172 4 28 138 143 143 152 155 159 159 162 164 167 173 169 175 182 4 29 126 129 130 137 139 141 143 147 151 150 156 158 160 166 4 30 133 136 136 144 149 148 150 154 156 160 165 165 167 168 4 31 139 142 140 148 151 152 153 156 156 157 164 163 164 166 4 32 119 125 125 131 134 136 137 138 141 142 147 147 149 152

APPENDIX 2 Mucositis Scores Ani- DAYS Group mal 6 8 10 12 14 16 18 20 22 24 26 28 1 1 0 1 1 1 2 3 4 4 3 2 2 1 1 2 0 1 1 1 2 3 4 3 3 3 2 1 1 3 0 1 2 2 3 3 4 3 2 2 2 1 1 4 1 1 1 2 2 3 4 3 3 3 2 1 1 5 0 1 1 2 2 3 4 3 2 2 1 1 1 6 0 0 1 2 2 3 4 3 3 3 3 1 1 7 0 0 1 3 2 3 4 3 3 2 1 1 1 8 0 0 1 2 3 4 4 3 2 2 1 1 2 9 0 1 2 2 3 4 4 3 3 3 2 0 2 10 1 1 1 2 2 3 4 3 3 3 3 1 2 11 0 1 1 2 3 3 4 3 3 2 2 1 2 12 0 0 2 3 3 3 4 3 2 3 2 1 2 13 1 1 3 2 3 3 3 2 2 2 2 1 2 14 0 1 1 2 2 3 4 3 3 3 2 1 2 15 0 1 1 1 2 3 4 4 2 2 2 1 2 16 0 1 1 2 2 3 4 3 2 2 1 1 3 17 1 1 1 2 2 3 4 3 2 2 2 1 3 18 0 0 1 2 3 3 4 3 2 2 2 2 3 19 1 1 1 2 3 3 4 3 2 3 2 1 3 20 1 1 1 2 2 3 3 2 1 2 1 1 3 21 1 1 1 2 2 3 4 3 3 3 3 1 3 22 0 1 1 1 2 3 4 3 2 2 2 1 3 23 0 1 1 2 2 3 4 3 1 2 2 1 3 24 1 1 1 2 2 3 3 3 2 2 1 0 4 25 0 1 1 1 2 3 4 4 4 3 3 1 4 26 0 1 1 2 2 3 4 3 3 2 2 1 4 27 0 0 2 2 2 3 4 3 3 2 3 2 4 28 1 1 1 2 2 3 3 3 2 2 3 3 4 29 0 0 1 2 3 3 4 3 2 2 1 1 4 30 1 1 1 2 2 2 2 1 2 2 1 1 4 31 0 0 1 2 2 3 3 2 1 2 1 1 4 32 1 0 1 2 3 4 4 3 3 2 1 1

Example 2 A Second Study of SCV-07 in the Treatment of Oral Mucositis Induced by Acute Radiation in Hamsters

The objective of this study was to conduct a preliminary evaluation of the efficacy of SCV-07 in the treatment of oral mucositis using an acute radiation induced hamster model of the disease. SCV-07 was given at doses of 1 mg/kg or 100 μg/kg once or twice daily by sub-cutaneous injection for 22 days, starting one day before radiation and continuing until day 20 after radiation. No deaths were observed in any of the treatment groups and there were statistically significant increases in growth rate, suggesting that SCV-07 was well tolerated at these doses, and may actually reduce the weight loss associated with mucositis. Control hamsters had a mucositis score of 3 or higher on 28.1% of the animal days evaluated in this study. Animals treated with SCV-07 at 100 μg/kg once daily on days −1 to 20 showed a statistically significant reduction in the number of animals days with a score of 3 or higher to 6.3% (P<0.001), and a statistically significant reduction in individual daily scores on days 14 (P=0.011), 16 (P=0.002) and 18 (P=0.001). Animals treated with SCV-07 at 100 μg/kg twice daily on days −1 to 20 showed a statistically significant reduction in the number of animals days with a score of 3 or higher to 8.9% (P<0.001), and a statistically significant reduction in individual daily scores on days 18 (P<0.001) and 20 (P=0.003). Animals treated with SCV-07 at 1 mg/kg twice daily on days −1 to 20 showed a statistically significant reduction in the number of animals days with a score of 3 or higher to 12.5% (P<0.001), and a statistically significant reduction in individual daily scores on days 16 (P=0.043) 18 (P=0.009) and 20 (P=0.007). Animals treated with SCV-07 at 1 mg/kg twice daily on days −1 to 20 did not show any significant reduction in mucositis scores. There was no statistically significant difference in the number of animal days with a score of 3 or higher between the group treated with SCV-07 at 100 μg/kg once daily and the group treated with SCV-07 at 1 mg/kg twice daily (P=0.054). When individual daily scores were compared between these two groups, a single day of significant difference was observed on day 14 (P=0.005). These observations suggest that treatment with SCV-07 at 100 μg/kg once daily is very close to being significantly better that treatment with SCV-07 at 1 mg/kg twice daily. This observation suggests that 100 μg/kg once daily is the dose closest to the optimal dose of the doses tested in this study and in the prior study SCI-01.

Introduction 1.1 Background

Oral ulcerative mucositis is a common, painful, dose-limiting toxicity of drug and radiation therapy for cancer. The disorder is characterized by breakdown of the oral mucosa that results in the formation of ulcerative lesions. In granulocytopenic patients, the ulcerations that accompany mucositis are frequent portals of entry for indigenous oral bacteria often leading to sepsis or bacteremia. Mucositis occurs to some degree in more than one third of patients receiving anti-neoplastic drug therapy. The frequency and severity are significantly greater among patients who are treated with induction therapy for leukemia or with many of the conditioning regimens for bone marrow transplant. Among these individuals, moderate to severe mucositis is not unusual in more than three-quarters of patients. Moderate to severe mucositis occurs in virtually all patients who receive radiation therapy for tumors of the head and neck and typically begins with cumulative exposures of 15 Gy and then worsens as total doses of 60 Gy or more are reached.

Clinically mucositis progresses through three stages:

1. Inflammation accompanied by painful mucosal erythema, which can respond to local anesthetics. 2. Painful ulceration with pseudomembrane formation and, in the case of myelosuppressive treatment, potentially life-threatening sepsis, requiring antimicrobial therapy. Pain is often of such intensity as to require parenteral narcotic analgesia. 3. Spontaneous healing, occurring about 2-3 weeks after cessation of anti-neoplastic therapy.

Standard therapy for mucositis is predominantly palliative, including application of topical analgesics such as lidocaine and/or systemic administration of narcotics and antibiotics. Currently, there is only one approved treatment for oral mucositis, Kepivance (Palifermin), which is only approved for the treatment of oral mucositis in patients undergoing conditioning regimens prior to hematopoetic stem cell transplantation for the treatment of hematologic malignancies.

The complexity of mucositis as a biological process has only been recently appreciated. It has been suggested that the condition represents a sequential interaction of oral mucosal cells and tissues, reactive oxygen species, pro-inflammatory cytokines, mediators of apoptosis and local factors such as saliva and the oral micro biota. While epithelial degeneration and breakdown ultimately result in mucosal ulceration, it appears that the early changes associated with radiation-induced mucosal toxicity occur within the endothelium, and connective tissue of the submucosa. Electron microscopic evaluation of mucosa within 1 week of radiation shows damage to both endothelium and connective tissue, but not epithelium. Such injury is likely mediated by free radical formation. It appears that the overall mechanism for mucositis development is similar for both radiation and chemotherapy.

1.2 Acute Radiation Model

The acute radiation model in hamsters, developed by the Principle Investigator, has proven to be an accurate, efficient and cost-effective technique to provide a preliminary evaluation of anti-mucositis compounds. The course of mucositis in this model is well defined and results in peak scores approximately 14-16 Days following radiation. The acute model has little systemic toxicity, resulting in few hamster deaths, thus permitting the use of smaller groups (N=7-8) for initial efficacy studies. It has also been used to study specific mechanistic elements in the pathogenesis of mucositis. Molecules that show efficacy in the acute radiation model may be further evaluated in the more complex models of fractionated radiation, chemotherapy, or concomitant therapy.

In this study, an acute radiation dose of 40 Gy on day 0 was administered. Clinically significant mucositis was observed on days 12 through 28.

2. Study Objective and Summary 2.1 Study Objective

The objective of this study was to evaluate the effect of SCV-07, administered by sub-cutaneous injection on different schedules between the day before radiation and the twentieth day after radiation, on the frequency, severity and duration of oral mucositis induced by acute radiation. A previous study with SCV-07 (Study SCI-01, Example 1), had indicated some activity against mucositis at a dose of 100 μg/kg, given once daily from day −1 to day 16. In this study, SCV-07 was dosed at 100 μg/kg and 1 mg/kg once or twice daily from day −1 to day 20, to see if the partial effect seen in the previous study could be extended.

2.2 Study Summary

Forty (40) Syrian Golden Hamsters were given an acute radiation dose of 40 Gy directed to their left buccal cheek pouch on day 0. Test material SCV-07 was given by sub-cutaneous injection at 100 μg/kg or 1 mg/kg once or twice daily. Dosing began one day before radiation (day −1) and continued until day 20. Mucositis was evaluated clinically starting on day 6, and continuing on alternate days until day 28.

3. Evaluation 3.1 Mucositis Evaluation

The grade of mucositis was scored, beginning day 6, and for every second day thereafter, through and including day 28. The effect on mucositis of each drug treatment compared to placebo was assessed according to the following parameters:

3.1.1 The Difference in the Number of Days Hamsters in Each Group have Ulcerative (score ≧3) mucositis.

On each evaluation day, the number of animals with a blinded mucositis score of ≧3 in each drug treatment group was compared to the control group. Differences were compared on a cumulative basis and statistical significance was determined by chi-square analysis. Efficacy, in this analysis, is defined by a significant reduction in the number of days that a group of animals had ulcerations (scores ≧3) when compared to the control group. This same test was also used to evaluate differences between different drug treatment groups.

3.1.2 Rank Sum Differences in Daily Mucositis Scores.

For each evaluation day the scores of the control group were compared to those of the treated groups using non-parametric rank sum analysis. Treatment success was considered as a statistically significant lowering of scores in the treated group on 2 or more days from day 6 to day 28.

3.2 Weights and Survival

All animals were weighed daily and their survival recorded, in order to assess possible differences in animal weight among treatment groups as an indication for mucositis severity and/or possible toxicity resulting from the treatments.

4. Study Design

Forty Syrian Golden Hamsters were divided into five (5) groups of eight (8) animals each. All animals received a single dose of acute radiation of 40 Gy directed to their left buccal cheek pouch on day 0. This was accomplished by anesthetizing the animals and everting the left buccal pouch, while protecting the rest of the animals with a lead shield. Test materials were given by sub-cutaneous injection once daily as detailed in Table 5. Mucositis was evaluated clinically starting on Day 6, and continuing on alternate days until day 28.

TABLE 5 SCI-02. Study Design Group Number of Treatment Volume Number Animals Treatment Schedule* (mL) 1 8 males Vehicle (PBS), sc, qd Day −1 to 20 Adjust per body weight 2 8 males SCV-07, sc, qd Day −1 to 20 Adjust per 100 μg/kg, body weight 3 8 males SCV-07, sc, bid Day −1 to 20 Adjust per 100 μg/kg, body weight 4 8 males SCV-07, sc, qd Day −1 to 20 Adjust per 1.0 mg/kg body weight 5 8 males SCV-07, sc, bid Day −1 to 20 Adjust per 1.0 mg/kg body weight *The dose on day 0 was performed 30 minutes prior to radiation

5. Material and Methods 5.1 Location of Study Performance

The study was performed at Biomodels AAALAC accredited facility in Watertown Mass. Approval for this study was obtained from Biomodels IACUC.

5.2 Animals

Male LVG Syrian Golden Hamsters (Charles River Laboratories), aged 5 to 6 weeks, with average body weight of 81.9 g at study commencement, were used. Animals were individually numbered using an ear punch and housed in small groups of 8 animals per cage. Animals were acclimatized prior to study commencement. During this period of 5 days, the animals were observed daily in order to reject animals that presented in poor condition.

5.3 Housing

The study was performed in animal rooms provided with filtered air at a temperature of 70° F.+/−5° F. and 50%+/−20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. Bed-O-Cobs® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity was recorded during the study, and the records retained.

5.4 Diet

Animals were fed with a Purina Labdiet® 5061 rodent diet and water was provided ad libitum.

5.5 Animal Randomization and Allocations.

Animals were randomly and prospectively divided into five (5) treatment groups prior to irradiation. Each animal was identified by an ear punch corresponding to an individual number. For more consistent identification, ear punch numbering was used rather than tagging, since tags may become dislodged during the course of the study. A cage card was used to identify each cage or label marked with the study number (SCI-02), treatment group number and animal numbers.

5.6 Sub-Cutaneous Dosing and Drug Application

The test compound, SCV-07 was provided as a powder and dissolved in sterile PBS immediately prior to administration. Three dilutions were prepared: 100 μg/mL, 10 μg/mL and 1 μg/mL. Drug was given in a volume of 0.1 mL per 100 g body weight, with the appropriate dilution of SCV-07 for each group, using a tuberculin syringe with a 27G needle. Injections were given subcutaneously to the back or abdomen.

5.7 Mucositis Induction

Mucositis was induced using a standardized acute radiation protocol. A single dose of radiation (40 Gy/dose) was administered to all animals on day 0. Radiation was generated with a 160 kilovolt potential (15-ma) source at a focal distance of 30 cm, hardened with a 0.35 mm Cu filtration system. Irradiation targeted the left buccal pouch mucosa at a rate of 3.2 Gy/minute. Prior to irradiation, animals were anesthetized with an intraperitoneal injection of Ketamine (160 mg/kg) and Xylazine (8 mg/kg). The left buccal pouch was everted, fixed and isolated using a lead shield to protect all other parts of the hamster's body.

5.8 Mucositis Scoring

The mucositis score, weight change and survival were measured throughout the study as described above. For the evaluation of mucositis, the animals were anesthetized with an inhalation anesthetic, and the left pouch everted. Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 for normal, to 5 for severe ulceration (clinical scoring). In descriptive terms, this scale is defined as follows:

TABLE 6 SCI-02: Mucositis Scoring. Score: Description: 0 Pouch completely healthy. No erythema or vasodilation. 1 Light to severe erythema and vasodilation. No erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of superficial aspects of mucosa leaving denuded areas. Decreased stippling of mucosa. 3 Formation of off-white ulcers in one or more places. Ulcers may have a yellow/gray due to pseudomembrane. Cumulative size of ulcers should equal about ¼ of the pouch. Severe erythema and vasodilation. 4 Cumulative seize of ulcers should equal about ½ of the pouch. Loss of pliability. Severe erythema and vasodilation. 5 Virtually all of pouch is ulcerated. Loss of pliability (pouch can only partially be extracted from mouth).

A score of 1-2 is considered to represent a mild stage of the disease, whereas a score of 3-5 is considered to indicate moderate to severe mucositis. Following visual scoring, a photograph was taken of each animal's mucosa using a standardized technique. At the conclusion of the experiment, all films were developed and the photographs randomly numbered. At least two independent trained observers graded the photographs in blinded fashion using the above-described scale (blinded scoring).

6. Results and discussion 6.1 Survival

No deaths were observed during the course of this study.

6.2 Weight Change

The mean daily percent weight change data was evaluated. The saline treated control hamsters gained an average of 44.1% of their starting weight during the study. Hamsters in the group receiving SCV-07 100 μg/kg once daily on days −1 to 20 gained an average of 49.9% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 100 μg/kg twice daily on days −1 to 20 gained an average of 61.3% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 1 mg/kg once daily on days −1 to 20 gained an average of 63.4% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 1 mg/kg twice daily on days −1 to 20 gained an average of 69.1% of their starting weight during the study. The significance of these differences was evaluated by calculating the area-under-the-curve (AUC) for the weight gain of each animal, and then comparing the different treatment groups using a One-Way ANOVA test. The results of this analysis indicated that there were significant differences between the SCV-07 treated groups and the control group (P=0.012). The groups treated with SCV-07 100 μg/kg twice daily, 1 mg/kg once and twice daily all had significantly greater weight gain than the saline controls (P=0.014, P=0.009 and P=0.004 respectively).

6.3 Mucositis (Tables 7 & 8)

Mean daily mucositis scores for each group are were determined. In the saline treated control group, peak levels of mucositis were seen on day 18, when the mean score reached 3.0. The group receiving SCV-07 at 100 μg/kg once daily from day −1 to 20 had a peak mean mucositis score of 2.2 on day 16. The group receiving SCV-07 at 100 μg/kg twice daily from day −1 to 20 had a peak mean mucositis score of 2.5 which occurred on days 14 and 16. The group receiving SCV-07 at 1 mg/kg once daily from day −1 to 20 had a peak mean mucositis score of 2.9 on day 14. The group receiving SCV-07 at 1 mg/kg twice daily from day −1 to 20 had a peak mean mucositis score of 2.6 on day 14. The significance of the differences observed between the different treatment groups was evaluated by calculating the number of days with a score of 3 or higher for each group and comparing these numbers using a chi-squared (□²) test. The results of this analysis are shown in Table 7. The hamsters in the saline treated control group had a score of 3 or higher on 28.1% of the animal days evaluated. In the group receiving SCV-07 at 100 μg/kg once daily from day −1 to 20, a mucositis score of 3 or higher was observed on 6.3% of the animals days evaluated, which was statistically significantly different that the controls (P<0.001). The hamsters in the groups treated with SCV-07 at 100 μg/kg twice daily on days −1 to 20 had a score of 3 or higher on 8.9% of animal days respectively, which was statistically significantly different from the controls (P<0.001). The group treated with SCV-07 at 1 mg/kg once daily on days −1 to 20 had a score of 3 or higher on 28.1% of animal days, which was not significantly different from controls (P=1.000). The group treated with SCV-07 at 1 mg/kg twice daily on days −1 to 20 had a score of 3 or higher on 12.5% of animal days, which was significantly different from controls (P<0.001). There was no statistically significant difference in the number of animal days with a score of 3 or higher between the group treated with SCV-07 at 100 μg/kg once daily and the group treated with SCV-07 at 1 mg/kg twice daily (P=0.054).

A further analysis of the mucositis scores was performed using the Mann-Whitney rank sum analysis to compare the scores for each group on each day. The results of this analysis are shown in Table 8. In this analysis, 2 days of significant reduction in the mucositis score are generally required before it is regarded as meaningful reduction in mucositis. The group treated with SCV-07 at 100 μg/kg once daily on days −1 to 20 showed statistically significant improvement on days 14 (P=0.011), 16 (P=0.002) and 18 (P=0.001) of the study relative to the saline controls. The group treated with SCV-07 at 100 μg/kg twice daily on days −1 to 20 showed significant improvement relative to controls on days 18 (P<0.001) and 20 (P=0.003). The group treated with SCV-07 at 1 mg/kg once daily on days −1 to 20 showed no significant improvement in mucositis on any day of the study relative to controls. The group treated with SCV-07 at 1 mg/kg twice daily on days −1 to 20 showed significant improvement relative to controls on days 16 (P=0.043) 18 (P=0.009) and 20 (P=0.007). A comparison between the group treated with SCV-07 at 100 μg/kg once daily and the group treated with SCV-07 at 1 mg/kg twice daily showed a single day of statistically significant difference on day 14 (P=0.005).

TABLE 7 SCI-02. Chi-square analysis of the total number of days the animals in each group spent with a score of three or more. This statistic is a measure of severity of ulceration, a clinically important outcome. Days Days Total % Days Chi Sq Group >=3 <3 Days >=3 v control P Value Vehicle (PBS), qd, 54 138 192 28.1 — — sc SCV-07 12 180 192 6.3 27.828 <0.001 100 ug/kg, qd, sc SCV-07 17 175 192 8.9 21.4010 <0.001 100 ug/kg, bid, sc SCV-07 54 138 192 28.1 0.0000 1.000 1 mg/kg, qd, sc SCV-07 24 168 192 12.5 12.7360 <0.001 1 mg/kg, bid, sc

TABLE 8 SCI-02. The significance of group differences observed in daily mucositis scores was determined using the Mann-Whitney rank sum test. This nonparametric statistic is appropriate for the visual mucositis scoring scale. The p values for each calculation are shown. Significant improvements are shown underlined. Day Group Comparison 6 8 10 12 14 16 18 20 22 24 26 28 Control vs 0.558 0.985 0.985 0.425 0.011 0.002 0.001 0.092 0.413 0.436 0.555 0.374 SCV-7 100 ug/kg, qd, sc Days −1 to 20 Control vs 0.558 0.554 0.985 0.581 0.664 0.204 <0.001  0.003 0.279 0.805 0.775 0.558 SCV-7 100 ug/kg, bid, sc Days −1 to 20 Control vs 0.233 0.985 0.985 0.775 0.218 0.662 0.116 0.405 0.404 0.298 0.774 0.895 SCV-7 1 mg/kg, qd, sc Days −1 to 20 Control vs 0.072 0.985 0.985 0.805 0.925 0.043 0.009 0.007 0.581 0.985 0.774 0.777 SCV-7 1 mg/kg, bid, sc Days −1 to 20 SCV-7 100 ug/kg, qd, sc vs 0.233 0.985 0.985 0.279 0.005 0.232 0.335 0.371 0.775 0.370 0.370 0.557 SCV-7 1 mg/kg, bid, sc Days −1 to 20

7. Conclusions

1. There was no evidence of any toxicity from SCV-07 in this study based on the observations of mortality and weight gain. 2. The groups treated with SCV-07 at 100 μg/ml twice daily or SCV-07 1 mg/ml once or twice daily showed significant increases in weight gain relative to the saline controls (P=0.014, P=0.009 and P=0.004 respectively). 3. Animals treated with SCV-07 at 100 μg/kg once daily on days −1 to 20 showed a statistically significant reduction in the number of animals days with a score of 3 or higher (P<0.001), and a statistically significant reduction in individual daily scores on days 14 (P=0.011), 16 (P=0.002) and 18 (P=0.001). 4. Animals treated with SCV-07 at 100 μg/kg twice daily on days −1 to 20 showed a statistically significant reduction in the number of animals days with a score of 3 or higher (P<0.001), and a statistically significant reduction in individual daily scores on days 18 (P<0.001) and 20 (P=0.003). 5. Animals treated with SCV-07 at 1 mg/kg twice daily on days −1 to 20 showed a statistically significant reduction in the number of animals days with a score of 3 or higher (P<0.001), and a statistically significant reduction in individual daily scores on days 16 (P=0.043) 18 (P=0.009) and 20 (P=0.007). 6. Animals treated with SCV-07 at 1 mg/kg twice daily on days −1 to 20 did not show any significant reduction in mucositis scores. 7. There was no statistically significant difference in the number of animal days with a score of 3 or higher between the group treated with SCV-07 at 100 μg/kg once daily and the group treated with SCV-07 at 1 mg/kg twice daily (P=0.054). When individual daily scores were compared between these two groups, a single day of significant difference was observed on day 14 (P=0.005).

These observations suggest that treatment with SCV-07 at 100 μg/kg once daily is very close to being significantly better that treatment with SCV-07 at 1 mg/kg twice daily.

9. Appendices

APPENDIX 3 Animal Weights Group Animal 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 1 84.7 84.6 87 89 92 94 96 98 98 102 104 105 107 104 106 105 1 2 79.1 79 79 83 88 88 92 91 93 101 101 102 104 105 108 108 1 3 84.1 84.3 86 88 93 93 95 97 97 102 104 105 115 105 105 103 1 4 81.3 79.1 81 82 84 84 86 89 90 92 90 96 99 97 99 100 1 5 84.6 86.9 86 89 93 95 98 100 99 106 108 109 110 109 108 108 1 6 90.9 92.3 94 96 99 103 107 108 107 113 113 113 105 113 117 116 1 7 80.3 78.5 82 83 86 89 91 92 95 97 100 100 103 99 101 101 1 8 85.3 85.2 89 90 95 98 99 100 105 108 108 108 109 109 111 110 2 9 87.6 86.5 92 96 98 102 105 107 108 114 117 109 120 118 124 123 2 10 87.2 80.5 86 94 97 99 101 103 105 111 110 103 114 110 116 116 2 11 81.6 81.7 86 86 87 88 90 90 94 95 97 90 98 95 98 99 2 12 71.9 72.8 75 77 79 79 85 84 85 88 91 88 97 97 101 98 2 13 86.2 85.7 91 93 94 95 98 97 99 101 105 98 109 104 110 110 2 14 81.9 82.6 86 87 90 90 92 94 94 101 100 95 104 102 106 102 2 15 82.7 83.4 88 91 93 95 97 99 100 105 106 101 109 106 110 111 2 16 80.4 81.2 90 88 89 92 93 94 98 98 103 99 107 106 111 113 3 17 77.1 77.1 80 83 79 84 91 91 85 98 100 99 101 97 103 104 3 18 75 75.8 82 81 81 82 85 85 81 90 92 91 92 88 94 94 3 19 86.6 86.1 93 94 92 98 102 105 99 113 115 117 120 116 121 127 3 20 80.2 79.1 86 89 84 86 93 95 89 100 103 102 104 101 105 106 3 21 81.7 81 87 90 85 89 92 94 87 98 100 100 104 99 105 104 3 22 82 80.3 86 89 85 89 92 95 88 99 102 102 104 100 106 105 3 23 80.8 80.8 87 90 89 93 101 100 93 107 107 108 115 108 113 114 3 24 83 80.7 88 94 85 94 102 102 95 110 113 113 118 117 120 121 4 25 85.2 84.3 86 89 93 92 98 98 100 106 105 107 109 108 110 107 4 26 88.7 89.4 94 98 100 101 104 107 108 113 114 116 119 118 121 120 4 27 83.4 84.9 86 90 92 93 97 97 98 102 103 105 105 105 108 105 4 28 72.4 75.7 77 79 81 81 85 85 86 90 92 93 94 94 93 91 4 29 72.3 75.8 77 78 83 82 87 91 91 99 99 102 103 104 105 106 4 30 90.6 94 96 99 101 104 109 111 109 115 116 115 103 118 120 120 4 31 75.9 77.5 79 82 84 85 89 91 91 98 99 100 117 103 105 105 4 32 84.8 86.4 91 95 99 98 103 104 102 109 111 111 116 116 118 116 5 33 90.1 87.6 89 93 94 98 106 106 107 112 113 113 117 118 119 118 5 34 76.5 77.3 78 79 81 81 83 86 87 90 91 90 92 93 117 99 5 35 87.5 89.6 91 95 98 98 105 106 109 114 116 118 122 119 95 120 5 36 81 82.8 84 89 96 93 101 100 101 106 107 107 110 113 122 114 5 37 74.7 74.2 83 79 81 85 87 88 91 95 97 98 99 99 100 99 5 38 79.8 81.8 81 87 90 90 94 96 98 104 104 105 109 108 112 112 5 39 77.3 76.6 75 85 87 88 90 92 94 97 99 99 102 103 106 107 5 40 80.1 78.8 80 83 86 86 95 96 97 99 102 100 102 102 104 104 Group Animal 16 17 18 19 20 21 22 23 24 25 26 27 28 1 1 109 104 102 109 110 113 113 117 118 119 118 118 116 1 2 112 106 108 116 115 122 122 127 129 129 128 132 126 1 3 107 101 99 107 108 111 111 114 116 119 117 118 114 1 4 105 99 97 105 104 108 125 112 112 114 114 115 111 1 5 112 106 107 113 113 118 121 121 123 125 124 123 123 1 6 120 112 111 120 119 127 127 132 134 134 133 134 132 1 7 105 97 95 105 107 111 113 118 117 121 118 121 120 1 8 1131 107 106 111 118 117 117 121 123 125 123 129 123 2 9 130 122 123 130 124 124 135 142 135 142 137 136 142 2 10 121 117 110 124 117 117 134 130 124 131 127 125 131 2 11 104 98 107 105 98 98 100 108 102 108 116 101 107 2 12 108 101 100 108 102 102 112 120 112 120 104 114 120 2 13 115 109 108 115 108 107 115 122 116 120 118 114 121 2 14 105 100 98 109 103 103 110 118 110 117 115 110 117 2 15 118 111 95 119 111 110 120 128 121 119 118 113 118 2 16 118 117 114 120 114 114 121 128 126 128 129 124 131 3 17 107 108 111 111 114 117 110 122 115 126 122 129 127 3 18 98 100 101 102 105 108 105 110 112 114 111 117 117 3 19 134 133 138 136 140 141 136 145 147 150 145 153 151 3 20 113 110 115 112 114 116 112 117 119 121 119 123 120 3 21 109 106 109 109 110 112 105 114 120 116 114 119 118 3 22 110 112 114 114 118 118 112 122 124 124 122 129 126 3 23 119 119 123 123 126 129 120 134 136 137 135 141 139 3 24 126 128 130 131 133 137 127 140 142 143 142 147 146 4 25 113 114 116 116 120 119 113 123 126 126 126 129 133 4 26 126 127 125 130 133 136 136 139 142 142 144 145 147 4 27 111 114 115 115 118 122 114 126 127 129 130 132 135 4 28 93 94 96 95 98 99 99 104 103 105 105 107 111 4 29 110 114 111 115 119 121 112 125 127 129 130 130 134 4 30 125 126 120 129 131 133 124 134 135 138 138 138 139 4 31 112 112 114 115 117 118 112 120 123 127 127 128 131 4 32 122 123 124 122 124 128 119 130 131 133 131 132 135 5 33 125 125 128 131 130 136 134 137 135 142 145 148 150 5 34 103 104 106 106 107 109 113 115 113 120 117 121 123 5 35 127 127 129 130 130 133 133 137 135 141 141 144 147 5 36 120 122 125 124 127 132 131 136 135 140 141 144 147 5 37 105 104 106 107 109 111 110 115 112 118 120 124 126 5 38 115 118 120 121 121 124 124 129 129 132 133 137 139 5 39 112 113 115 115 117 120 120 125 127 131 132 133 137 5 40 108 107 109 111 111 114 113 117 116 121 123 124 125

APPENDIX 4 Mucositis Scores Ani- DAY Group mal 6 8 10 12 14 16 18 20 22 24 26 28 1 1 0 1 2 2 2 3 4 3 2 2 2 2 1 1 0 1 1 2 2 3 4 3 2 2 2 1 1 2 0 1 2 3 3 3 3 2 2 1 1 1 1 2 0 1 1 3 3 3 3 2 2 2 2 1 1 3 0 1 2 2 3 3 3 3 2 2 2 2 1 3 0 1 1 2 3 3 3 3 2 2 2 1 1 4 0 1 2 2 1 1 2 1 1 1 2 2 1 4 0 1 1 1 2 1 2 2 2 2 2 1 1 5 0 1 2 2 3 3 3 3 2 2 2 2 1 5 0 1 1 2 3 3 3 3 2 2 2 1 1 6 1 1 2 2 2 3 3 3 2 2 2 2 1 6 1 1 1 2 2 3 3 3 2 2 2 1 1 7 1 1 2 2 3 3 3 2 2 2 2 2 1 7 1 1 1 2 3 3 3 2 2 2 2 1 1 8 1 1 2 2 3 3 3 3 3 3 2 2 1 8 1 1 1 2 3 3 3 3 3 3 2 2 2 9 1 1 2 2 2 2 2 2 1 1 1 1 2 9 1 1 1 2 2 2 2 2 2 2 2 1 2 10 1 1 2 3 2 2 2 2 2 2 2 2 2 10 1 1 1 2 2 2 2 2 2 2 2 1 2 11 1 1 2 2 2 2 3 3 2 2 1 2 2 11 1 1 1 2 2 2 3 3 2 2 2 1 2 12 0 1 2 2 2 2 1 2 2 1 2 1 2 12 0 1 1 2 2 2 2 2 2 2 2 1 2 13 0 1 2 2 2 2 1 1 1 1 2 1 2 13 0 1 1 1 1 2 2 2 2 2 2 1 2 14 0 1 2 2 3 3 3 3 2 2 2 2 2 14 0 1 1 1 2 3 3 3 2 2 2 1 2 15 1 1 2 2 2 2 2 2 2 2 2 2 2 15 1 1 1 2 2 2 2 2 2 2 2 1 2 16 0 1 2 2 2 2 2 2 2 2 1 2 2 16 0 1 1 1 2 2 2 2 2 2 2 1 3 17 0 1 2 2 2 3 2 2 1 2 2 1 3 17 0 1 1 2 2 3 2 2 2 2 2 1 3 18 1 1 2 2 3 3 3 2 2 2 2 2 3 18 1 1 1 2 3 3 2 2 2 2 2 1 3 19 1 1 2 1 2 1 1 1 1 1 1 1 3 19 1 1 1 2 2 2 2 2 2 2 2 1 3 20 1 1 2 2 3 3 2 2 2 2 2 2 3 20 1 1 1 2 3 3 2 2 2 2 2 1 3 21 1 1 2 2 2 3 2 1 1 2 2 2 3 21 1 1 1 2 2 2 2 2 2 2 2 1 3 22 0 1 2 2 2 2 2 2 2 2 1 2 3 22 0 1 1 2 2 2 2 2 2 2 2 1 3 23 0 0 2 2 3 2 2 2 2 2 2 2 3 23 0 0 1 2 3 2 2 2 2 2 2 1 3 24 0 1 2 2 3 3 2 2 2 2 2 2 3 24 0 1 1 2 3 3 2 2 2 2 2 1 4 25 1 1 2 2 3 3 3 3 3 3 3 3 4 25 1 1 1 2 3 3 3 3 3 3 2 1 4 26 0 1 2 2 3 3 3 3 3 3 2 2 4 26 0 1 1 2 3 3 3 3 3 3 2 1 4 27 1 1 2 2 3 3 3 3 3 3 2 2 4 27 1 1 1 2 3 3 3 2 2 2 2 1 4 28 0 1 2 2 3 3 3 2 2 2 2 2 4 28 0 1 1 2 3 3 3 3 2 2 2 1 4 29 1 1 2 2 3 3 3 3 2 2 2 2 4 29 1 1 1 2 3 3 3 2 2 2 2 1 4 30 0 1 2 2 3 2 2 2 2 2 2 2 4 30 0 1 1 2 3 2 2 2 2 2 2 1 4 31 1 1 2 2 3 3 2 2 2 2 2 2 4 31 1 1 1 2 3 3 2 2 2 2 2 1 4 32 1 1 2 2 2 2 2 1 1 1 1 2 4 32 1 1 1 2 2 2 2 2 2 2 2 1 5 33 1 1 2 2 2 2 2 2 2 2 2 2 5 33 1 1 1 2 2 2 2 2 2 2 2 1 5 34 1 1 2 2 3 2 2 2 2 2 2 2 5 34 1 1 1 2 3 2 2 2 2 2 2 1 5 35 1 1 2 2 3 3 3 2 2 2 2 2 5 35 1 1 1 2 3 3 3 2 2 2 2 1 5 36 0 1 2 2 2 2 2 2 2 2 2 1 5 36 0 1 1 2 2 2 2 2 2 2 2 1 5 37 0 1 2 2 3 2 2 2 2 2 2 2 5 37 0 1 1 2 3 2 2 2 1 2 2 1 5 38 1 1 2 2 3 2 2 2 2 2 2 2 5 38 1 1 1 2 3 2 2 2 2 2 2 1 5 39 1 1 2 2 2 3 3 2 2 2 2 2 5 39 1 1 1 2 2 3 3 2 2 2 2 1 5 40 1 1 2 3 3 3 3 2 2 2 2 2 5 40 1 1 1 3 3 3 3 2 2 2 2 1

Example 3 A Study of the Efficacy of Effect of SCV-07 in the Treatment of Oral Mucositis Induced by Fractionated Radiation in Hamsters. 1. Introduction 1.1 Background

KGF-1 and other FGF family members have been shown to induce epithelial thickening of the oral and esophageal mucosal surfaces in BDF-1. SCV-07 and the derived SCV-07 peptide are believed to have mechanisms that that may overlap with KGF-1, and have been shown to be protective in other models of mucosal injury.

Oral ulcerative mucositis is a common, painful, dose-limiting toxicity of drug and radiation therapy for cancer. The disorder is characterized by breakdown of the oral mucosa that results in the formation of ulcerative lesions. In granulocytopenic patients, the ulcerations that accompany mucositis are frequent portals of entry for indigenous oral bacteria often leading to sepsis or bacteremia. Mucositis occurs to some degree in more than one third of patients receiving anti-neoplastic drug therapy. The frequency and severity are significantly greater among patients who are treated with induction therapy for leukemia or with many of the conditioning regimens for bone marrow transplant. Among these individuals, moderate to severe mucositis is not unusual in more than three-quarters of patients. Moderate to severe mucositis occurs in virtually all patients who receive radiation therapy for tumors of the head and neck and typically begins with cumulative exposures of 15 Gy and then worsens as total doses of 60 Gy or more are reached.

Clinically mucositis progresses through three stages:

1. Painful erythema which can generally be managed by topical anesthetics or non-narcotic analgesics . . . . 2. Painful ulceration often with pseudomembrane formation. In the case of concomitant myelosuppression, bacteremias or sepsis of oral origin are not uncommon. Pain is often of such intensity as to require narcotic analgesia, frequently parenterally. 3. Spontaneous healing, occurring about 2-3 weeks after cessation of anti-neoplastic therapy.

Currently, the only approved biologic or drug for mucositis prevention and/or treatment is Kepivance (palifermin). Kepivance use is limited to mucositis in patients receiving stem cell transplant for hematologic malignancies. Consequently, standard therapy for mucositis consists of palliative rinses, such as saline, bicarbonate solutions, mouthwashes, topical analgesics such as lidocaine and/or systemic administration of narcotics.

The complexity of mucositis as a biological process has only been recently appreciated. It has been suggested that the condition represents a sequential interaction of oral mucosal cells and tissues reactive oxygen species, pro-inflammatory cytokines, mediators of apoptosis, a range of signaling pathways, and local factors such as saliva and the oral micro biota. While epithelial degeneration and breakdown ultimately result in mucosal ulceration, it appears that the early changes associated with radiation-induced mucosal toxicity occur within the endothelium, and connective tissue of the submucosa. It appears that the overall mechanism for mucositis development is similar for both radiation and chemotherapy.

1.2 Fractionated Radiation Model

The fractionated radiation model in hamsters, developed by the Principal Investigator, has proven to be an accurate, efficient and cost-effective technique to provide a preliminary evaluation of anti-mucositis compounds. In this model, hamsters receive 8 doses of 7.5 Gy to the left cheek pouch on days 0, 1, 2, 3, 7, 8, 9 and 10, rather than the single dose of 40 Gy on day 0 that is used in acute radiation studies. The rationale for using this scheduling for the radiation is that it more closely resembles the clinical courses of radiotherapy given to cancer patients. The course of mucositis in this model is well defined and results in peak mucositis scores approximately 14-16 days following radiation. Mortality (generally from the side effects of anesthesia) in the fractionated radiation model is slightly higher than that seen in the acute radiation model and group sizes are increased (to 10 per group) to allow for this.

2. Study Objective and Summary 2.1 Study Objective

The objective of this study was to evaluate the effect of SCV-07, administered by sub-cutaneous injection, on the frequency, severity and duration of oral mucositis induced by a fractionated radiation protocol.

2.2 Study Summary

Forty (40) male Syrian Golden Hamsters were given eight doses of radiation of 7.5 Gy each directed to their left buccal cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9. This was accomplished by anesthetizing the animals and everting the left buccal pouch, while protecting the rest of the animal's bodies with a lead shield. Test materials were given by sub-cutaneous injection once daily as detailed in Table 9. Mucositis was evaluated clinically starting on day 7, and continuing on alternate days until day 35. Test articles were given as outlined Table 9, Groups 1 and 2 were dosed from Day −1 to Day 29, Group 3 was dosed on the days of radiation only, Group 4 was dosed on the days between Day −15 and Day 29 on which no radiation is given (Day −1, Day 4, Day 5 and Day 10 to Day 29).

3. Evaluation 3.1 Mucositis Evaluation

The grade of mucositis was scored, beginning day 6, and for every second day thereafter, through and including day 34. The effect on mucositis of each drug treatment compared to placebo was assessed according to the following parameters:

3.1.1 The Difference in the Number of Days Hamsters in Each Group have Ulcerative (Score ≧3) Mucositis.

On each evaluation day, the number of animals with a blinded mucositis score of ≧3 in each drug treatment group was compared to the control group. Differences were compared on a cumulative basis and statistical significance was determined by chi-square analysis. Efficacy, in this analysis, is defined by a significant reduction in the number of days that a group of animals had ulcerations (scores ≧3) when compared to the control group.

3.1.2 Rank Sum Differences in Daily Mucositis Scores.

For each evaluation day the scores of the control group were compared to those of the treated groups using non-parametric rank sum analysis. Treatment success was considered as a statistically significant lowering of scores in the treated group on 2 or more days from day 6 to day 28.

3.2 Weights and Survival

All animals were weighed daily and their survival recorded, in order to assess possible differences in animal weight among treatment groups as an indication for mucositis severity and/or possible toxicity resulting from the treatments.

4. Study Design

Forty (40) male Syrian Golden Hamsters were given eight doses of radiation of 7.5 Gy each directed to their left buccal cheek pouch on days 0, 1, 2, 3, 6, 7, 8 and 9. This was accomplished by anesthetizing the animals and everting the left buccal pouch, while protecting the rest of the animal's bodies with a lead shield. Test materials were given by sub-cutaneous injection once daily at 8:00 am as detailed in Table 9. Mucositis was evaluated clinically starting on Day 6, and continuing on alternate days until day 34. Test articles were given as outlined Table 9, Groups 1 and 2 was dosed from Day −1 to Day 29, Group 3 was dosed on the days of radiation only (days 0-3 and days 6-10), Group 4 was dosed on the days between Day −1 and Day 29 on which no radiation is given (Day −1, Day 4 to Day 5 and Day 10 to Day 29).

TABLE 9 SCI-03. Study Design Number Number Group of Treatment Treatment of Number Animals Schedule Doses 1 10 males Vehicle Control Day −1 to Day 29 31 2 10 males SCV-07 100 μg/kg Day −1 to Day 29 31 3 10 males SCV-07 100 μg/kg Day 0 to Day 3 8 and Day 6 to Day 9 4 10 males SCV-07 100 μg/kg Day −1, Days 4, 5 23 Day 10 to Day 29 Injections of SCV-07 on Days 0, 1, 2, 3, 6, 7, 8 and 9 were given approximately 30 minutes prior to radiation.

5. Material and Methods 5.1 Location of Study Performance

The study was performed at Biomodels AAALAC-accredited facility in Watertown, Mass.

5.2 Animals

Male LVG Syrian Golden Hamsters (Charles River Laboratories), aged 5 to 6 weeks, with average body weight of 81.0 g at study commencement, were used. Animals were individually numbered using an ear punch and housed in small groups of approximately 10 animals per cage. Animals were acclimatized for 5 days prior to study commencement and during this period, the animals were observed daily in order to reject animals that present in poor condition.

5.3 Housing

The study was performed in animal rooms provided with filtered air at a temperature of 70° F.+/−5° F. and 50%+/−20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. Bed-O-Cobs® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity was recorded during the study, and the records retained.

5.4 Diet

Animals were fed with a Purina Labdiet® 5061 rodent diet and water was provided ad libitum.

5.5 Animal Randomization and Allocations.

Animals were randomly and prospectively divided into four (4) treatment groups prior to irradiation. Each animal was identified by an ear punch corresponding to an individual number. For more consistent identification, ear punch numbering was used rather than tagging, since tags may become dislodged during the course of the study. A cage card was used to identify each cage or label marked with the study number (SCI-03), treatment group number and animal numbers.

5.6 Sub-Cutaneous Dosing and Drug Application

The test compound, human SCV-07 peptide was provided as a powder and dissolved in sterile PBS immediately prior to administration. Drug was given in a volume of 0.1, using a tuberculin syringe with a 27G needle. Injections were given subcutaneously to the back or abdomen.

5.7 Mucositis Induction

Radiation was generated with a Philips 160 kVp (kilovolt potential) (18.75-ma) X-ray source at a focal distance of 30 cm, with a 3.0 mm hardened Al filtration system. Irradiation was targeted to the left buccal pouch mucosa at a rate of 3.32 Gy/minute. Calibration of this source with a Victoreen model 530 dosimeter indicated that the dose rate was 28.57 nC/min. Using this calibration, the energy received by each animal at each radiation dose was approximately 64.5 nC (nanoCoulombs) at each time point. Prior to irradiation, animals were anesthetized with an intraperitoneal injection of ketamine (160 mg/kg) and xylazine (8 mg/kg). The left buccal pouch was everted, fixed and isolated using a lead shield.

5.8 Mucositis Scoring

The mucositis score, weight change and survival were measured throughout the study as described above. For the evaluation of mucositis, the animals were anesthetized with an inhalation anesthetic, and the left pouch everted. Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 for normal, to 5 for severe ulceration (clinical scoring). In descriptive terms, this scale is defined as follows:

TABLE 10 SCI-03: Mucositis Scoring. Score: Description: 0 Pouch completely healthy. No erythema or vasodilation. 1 Light to severe erythema and vasodilation. No erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of superficial aspects of mucosa leaving denuded areas, but no frank ulceration. Decreased stippling of mucosa. 3 Formation of off-white ulcers in one or more places. Ulcers may have a yellow/gray due to pseudomembrane. Cumulative size of ulcers should equal about ¼ of the pouch. Severe erythema and vasodilation. 4 Cumulative seize of ulcers should equal about ½ of the pouch. Loss of pliability. Severe erythema and vasodilation. 5 Virtually all of pouch is ulcerated. Loss of pliability (pouch can only partially be extracted from mouth).

A score of 1-2 is considered to represent a mild stage of the disease, whereas a score of 3-5 is considered to indicate moderate to severe mucositis. Following visual scoring, a photograph was taken of each animal's mucosa using a standardized technique. At the conclusion of the experiment, all films were developed and the photographs randomly numbered. At least two independent trained observers graded the photographs in blinded fashion using the above-described scale (blinded scoring).

6. Results 6.1 Survival

One death occurred during this study in the control group on day 8 as a consequence of anesthesia for radiation.

6.2 Weight Change

There were no significant differences in weight changes between study groups. The mean daily percent weight change data was evaluated. The saline treated control hamsters gained an average of 76.3% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 100 μg/kg on days −1 to 29 gained an average of 80.7% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 100 μg/kg on days of radiation only gained an average of 66.3% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 100 μg/kg on day −1, day 4, day 5 and days 10-29 gained an average of 69.7% of their starting weight during the study. The significance of these differences was evaluated by calculating the area-under-the-curve (AUC) for the weight gain of each animal, and then comparing the different treatment groups using a One-Way ANOVA test. The results of this analysis indicated that there were no significant differences between the different treatment groups (P=0.490).

6.3 Mucositis (Tables 11 & 12)

The kinetics and severity of mucositis development among control animals was consistent with that which was expected.

Mean daily mucositis scores for each group were evaluated. In the saline treated control group, the mean peak mucositis score was 3.2, which occurred on day 19. The group receiving SCV-07 from day −1 to day 29 had a peak mean mucositis score of 3.3, which occurred on day 19. The group receiving SCV-07 on the days of radiation (0-3 and 6-9) had a peak mean mucositis score of 3.0, which occurred on day 17. The group receiving SCV-07 on days 1, 4, 5 and 10 through 29 had a peak mean mucositis score of 2.9, which occurred on days 17, 19 and 23. The significance of the differences observed between the different treatment groups was evaluated by calculating the number of days with a score of 3 or higher for each group and comparing these numbers using a chi-squared (□²) test. The results of this analysis are shown in Table 11. The hamsters in the saline treated control group had a score of 3 or higher on 36% of the animal days evaluated. In the group receiving SCV-07 from day −1 to day 29, a mucositis score of 3 or higher was observed on 32.7% of the animals days evaluated, which was not statistically significantly different from controls (P=0.448). In the group receiving SCV-07 on days 0-3 and days 6-9, a mucositis score of 3 or higher was observed on 24% of the animals days evaluated, which was statistically significantly different from controls (P=0.002). In the group receiving SCV-07 on days −1, day 4 day 5 and day 10 to day 29, a mucositis score of 3 or higher was observed on 30.7% of the animals days evaluated, which was not statistically significantly different from controls (P=0.204). A further analysis of the mucositis scores was performed using the Mann-Whitney rank sum analysis to compare the scores for each group on each day. The results of this analysis are shown in Table 12. In this analysis, 2 days of significant reduction in the mucositis score are generally required before it is regarded as meaningful. The group treated with SCV-07 on days −1 to 29 was significantly better than the saline controls on days 29 (P=0.004), 31 (P=0.017) and 33 (P=0.002). The group treated with SCV-07 on days 0 to 3 and day 6 to day 9 was significantly better than the saline controls on days 21 (P=0.047), 23 (P<0.001), 25 (P=0.009), 29 (P<0.001), 31 (P=0.015) and 33 (P<0.001). The group treated with SCV-07 on days −1, 4, 5 and days 10 to 29 was significantly better than the saline controls on days 29 (P<0.001), 31 (P=0.004) and 33 (P<0.001).

TABLE 11 SCI-03. Chi-square analysis of the total number of days the animals in each group spent with a score of three or more. This statistic is a measure of severity of ulceration, a clinically important outcome. Days Days Total % Days Chi Sq Group >=3 <3 Days >=3 v control P Value Vehicle (PBS), qd, 98 174 272 36.0 — — sc Days −1 to 29 SCV-07 98 202 300 32.7 0.575 0.448 100 ug/kg, qd, sc Days −1 to 29 SCV-07 72 228 300 24.0 0.3160 0.002 100 ug/kg, qd, sc Days 0 to 3 and 6 to 9 SCV-07 92 208 300 30.7 0.2300 0.204 100 ug/kg, qd, sc Days −1, 4, 5 10 to 29

TABLE 12 SCI-03. The significance of group differences observed in daily mucositis scores was determined using the Mann-Whitney rank sum test. This nonparametric statistic is appropriate for the visual mucositis scoring scale. The p values for each calculation are shown. Significant reductions in mucositis scores relative to controls are shown underlined. Day Group Comparison 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 Control vs 0.989 0.295 0.526 0.297 0.490 0.988 0.608 0.129 0.062 0.781 0.526  0.004 0.017  0.002 0.872 SCV-7 100 ug/kg, qd, sc Days −1 to 29 Control vs 0.989 0.296 0.244 0.080 0.490 0.988 0.115 0.047 <0.001  0.009 0.224 <0.001 0.015 <0.001 0.872 SCV-7 100 ug/kg, qd, sc Days 0 to 3 and 6 to 9 Control vs 0.989 0.605 0.918 0.406 0.164 0.605 0.115 0.129 0.648 0.420 0.528 <0.001 0.004 <0.001 0.490 SCV-7 100 ug/kg, qd, sc Days −1, 4, 5 10 to 29

7. Conclusions

1. There was no evidence of any toxicity from SCV-07 in this study based on the observations of mortality and weight gain. 2. Animals treated with human SCV-07 at 100 μg/kg on days on which radiation was administered (days 0-3 and 6-9) showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P=0.002), and a significant reduction in mucositis scores on days 21 (P=0.047), 23 (P<0.001), 25 (P=0.009), 29 (P<0.001), 31 (P=0.015) and 33 (P<0.001). This result suggests that SCV-07 may be effective in reducing the overall severity of mucositis, which, consequently, results in enhanced resolution of mucosal injury. 3. Hamsters treated with SCV-07 on days −1 to 29 or on days on which radiation was not administered (days −1, 4, 5 and 10-29), did not show a significant reduction in the number of animal days with a score of 3 or higher, but did show significant reductions in mucositis scores on days 29, 31 and 33. 4. The contrast in effect of SCV-07 based on the schedule of radiation administration may provide some insight into its mechanism of action, but requires further evaluation. The fact that SCV-07 schedules in which the drug was administered on non-radiation days were not as efficacious is of interest, especially for animals treated on consecutive days. Furthermore, the observation that all animals treated with SCV-07 responded identically at the late stages of the study might suggest multiple effects of SCV-07 on the overall pathogenesis and resolution of radiation-induced mucositis.

9. Appendices

APPENDIX 5 Animal Weights Day Group Animal −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 1 82 87 86 88 89 89 94 95 98 97 100.8 103 105 108 109 110 110 112 115 1 2 78 82 81 84 87 85 91 94 95 Dead 1 3 79 79 77 76 75 75 77 77 79 80 82.2 85 85 89 88 87 86 86 89 1 4 88 92 93 96 98 99 103 107 108 107 113.9 116 116 120 121 123 122 124 125 1 5 77 80 79 84 88 86 91 93 90 88 96 96 96 97 98 98 98 100 104 1 6 83 85 90 91 95 94 90 100 100 101 106.1 107 109 112 113 114 115 115 118 1 7 76 78 78 79 81 83 98 88 90 89 93.4 95 93 96 98 98 99 99 102 1 8 73 76 75 76 77 78 84 86 86 87 90.5 93 97 98 99 101 103 102 103 1 9 81 86 87 88 87 90 94 99 98 100 104.4 105 108 111 113 113 114 114 119 1 10 79 83 84 82 84 80 88 90 91 90 93.7 95 95 97 96 97 95 100 102 2 11 82 86 89 78 77 80 83 88 88 89 94.5 95 98 100 103 104 104 105 108 2 12 80 82 82 83 86 85 88 92 91 90 93.4 92 95 96 97 96 97 99 100 2 13 82 84 84 87 88 88 92 94 93 94 98.3 100 99 103 104 106 105 107 108 2 14 73 77 76 79 79 82 84 87 87 87 90.6 92 96 97 97 98 99 99 101 2 15 82 87 89 92 95 95 98 102 102 103 108.5 108 110 114 115 116 117 117 120 2 16 83 85 88 91 94 93 97 102 99 99 103.5 105 107 111 111 109 108 109 111 2 17 81 82 83 87 88 87 91 95 93 95 99.7 97 101 104 106 105 107 106 107 2 18 76 77 76 80 79 79 84 86 85 87 88.2 92 92 95 94 95 94 96 97 2 19 84 87 87 88 90 91 94 98 100 98 102.3 104 104 107 108 108 108 111 110 2 20 69 70 74 76 75 76 79 79 84 84 88.6 90 94 97 98 98 98 99 103 3 21 87 92 92 97 94 95 100 104 106 103 110.6 111 112 102 114 113 112 113 118 3 22 76 78 79 80 80 82 85 89 90 88 94.6 95 96 110 101 100 100 103 106 3 23 93 98 95 102 104 102 108 111 115 113 117.4 119 122 125 124 125 126 129 132 3 24 78 82 79 80 82 81 85 88 89 88 92.6 93 95 97 98 99 97 97 100 3 25 80 84 82 85 84 86 88 90 91 93 94.4 96 99 101 103 100 102 101 104 3 26 87 89 86 88 86 86 89 91 89 90 93.7 95 97 98 101 100 101 100 101 3 27 84 87 87 88 89 88 93 97 97 97 101.7 103 104 103 109 112 111 113 115 3 28 80 82 83 84 88 85 88 94 93 94 97.5 98 99 99 105 103 105 106 106 3 29 79 79 78 81 83 83 87 89 90 88 91.6 94 94 96 96 97 96 99 102 3 30 76 76 73 74 76 74 77 78 78 78 79.2 80 81 84 84 84 82 85 86 4 31 80 84 85 89 90 93 94 96 97 98 101.2 102 104 105 107 105 106 109 111 4 32 85 88 90 91 94 94 99 102 103 105 110.3 110 112 116 116 116 116 117 117 4 33 82 85 82 96 87 85 89 92 93 91 95.3 95 96 97 97 96 96 96 96 4 34 87 89 87 90 86 91 97 99 101 103 108.6 110 110 115 117 118 120 122 123 4 35 84 89 90 92 97 97 104 107 110 108 111.7 114 115 117 119 121 122 124 127 4 36 85 90 89 91 94 96 98 101 103 102 104.7 108 107 109 110 110 111 112 113 4 37 87 91 91 92 94 95 98 101 102 102 106.7 108 109 112 113 113 113 113 114 4 38 89 94 92 97 98 99 106 106 109 106 110.6 112 114 115 117 117 119 121 122 4 39 70 73 72 73 73 73 76 79 78 79 80.7 80 78 82 81 83 83 86 87 4 40 81 87 86 85 88 87 87 90 92 89 90.6 89 90 93 88 88 89 94 97 Day Group Animal 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 1 1 115 117 119 120 122 124 125 126 130 130 132 132 135 137 137 141 143 144 1 2 Dead 1 3 90 95 95 97 98 101 101 102 106 108 107 108 111 114 115 119 118 120 1 4 126 129 130 128 130 133 131 131 134 135 138 136 138 138 139 143 142 143 1 5 105 106 109 113 114 117 120 121 126 129 129 128 134 135 137 142 143 142 1 6 120 123 126 128 130 132 138 138 144 147 146 147 152 155 155 160 159 159 1 7 103 109 111 111 111 116 118 118 124 123 125 125 130 131 133 137 138 137 1 8 103 110 109 110 112 115 115 117 121 122 123 121 126 129 129 133 131 134 1 9 120 124 128 126 129 132 132 134 138 139 140 140 145 146 147 151 155 153 1 10 105 105 109 110 110 113 114 115 118 119 121 120 122 124 125 129 131 133 2 11 108 113 114 116 117 120 122 123 129 131 134 132 136 138 141 146 145 146 2 12 98 104 105 107 108 111 113 113 117 116 118 118 122 124 126 131 130 131 2 13 109 114 116 117 119 120 124 124 129 130 130 130 137 138 141 136 145 136 2 14 103 107 109 110 112 114 116 117 121 124 124 123 128 131 120 145 136 145 2 15 120 124 126 127 127 130 132 132 137 138 139 139 142 143 143 147 146 147 2 16 113 116 117 122 123 126 128 128 132 133 135 134 138 142 142 148 147 148 2 17 118 122 121 126 127 130 130 132 136 137 140 140 144 146 147 152 153 154 2 18 98 102 103 104 105 108 111 110 114 115 114 114 120 121 122 125 125 127 2 19 113 115 116 119 120 120 123 126 129 130 133 133 137 139 139 144 147 146 2 20 106 108 111 118 117 118 120 121 124 126 131 127 133 138 138 144 146 147 3 21 117 122 123 124 127 127 128 128 131 133 134 132 135 138 139 143 143 144 3 22 109 112 114 115 114 121 122 121 124 125 127 126 130 132 133 136 136 137 3 23 137 138 141 141 144 152 148 150 153 158 158 158 162 165 165 172 171 170 3 24 101 105 107 108 108 113 114 115 119 119 122 122 126 128 128 133 134 135 3 25 104 108 109 108 112 113 115 116 117 119 121 120 122 123 124 127 127 128 3 26 102 106 108 109 111 113 115 116 122 119 121 122 125 128 130 133 133 135 3 27 108 111 112 114 115 118 120 121 124 124 125 126 129 131 131 132 134 134 3 28 108 113 114 114 118 120 121 122 120 126 128 128 132 134 134 140 138 140 3 29 104 107 107 109 111 114 115 114 119 122 120 121 125 125 128 131 131 132 3 30 87 90 90 93 93 96 95 95 97 97 100 99 102 104 103 107 107 110 4 31 112 118 118 120 124 124 123 125 126 129 131 131 133 136 138 141 140 141 4 32 114 120 121 124 124 127 128 131 134 134 136 134 138 142 142 146 147 146 4 33 95 98 99 101 102 105 107 108 110 114 114 112 116 118 119 122 122 124 4 34 122 128 128 131 133 135 138 139 142 143 145 146 150 152 153 158 157 158 4 35 127 132 133 136 134 137 138 140 143 144 146 144 149 151 152 157 158 159 4 36 115 119 118 120 122 123 124 128 128 131 131 131 136 136 138 141 140 141 4 37 113 118 118 119 120 120 123 122 123 127 128 128 132 134 134 137 139 140 4 38 121 127 128 131 133 135 135 137 139 140 142 142 146 150 149 154 153 154 4 39 88 92 92 94 95 97 99 98 100 102 103 102 105 108 109 112 112 113 4 40 98 102 104 109 109 111 112 113 118 121 122 118 126 126 129 132 133 134

APPENDIX 6 Mucositis Scores Day Number Group Animal 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 1 1 0 1 1 1 2 3 4 3 3 3 3 2 2 2 1 1 1 0 1 1 1 2 3 4 3 3 3 3 2 2 2 1 1 2 0 dead 1 2 0 1 3 0 1 1 2 3 3 4 3 3 2 2 2 2 2 1 1 3 0 1 1 2 3 3 4 3 3 2 2 2 2 2 1 1 4 0 1 1 2 3 3 3 3 3 2 2 1 1 2 1 1 4 0 1 1 2 3 3 3 3 3 2 2 1 1 2 1 1 5 0 1 2 2 3 3 3 3 2 2 2 2 2 2 1 1 5 0 1 2 2 3 3 3 3 2 2 2 2 2 2 1 1 6 0 1 1 2 3 3 3 3 3 3 2 2 2 2 1 1 6 0 1 1 2 3 3 3 3 3 3 2 2 2 2 1 1 7 0 1 1 2 3 3 3 3 3 2 2 1 1 1 0 1 7 0 1 1 2 3 3 3 3 3 2 2 1 1 1 0 1 8 0 1 1 1 2 3 3 4 4 3 2 2 1 1 0 1 8 0 1 1 1 2 3 3 4 4 3 2 2 1 1 0 1 9 0 1 1 1 2 3 3 3 3 2 2 1 0 1 0 1 9 0 1 1 1 2 3 3 3 3 2 2 1 0 1 0 1 10 0 1 2 2 3 3 3 3 3 3 3 3 3 2 1 1 10 0 1 2 2 3 3 3 3 3 3 3 3 3 2 1 2 11 0 1 1 2 3 3 3 3 3 3 3 3 2 2 1 2 11 0 1 1 2 3 3 3 3 3 3 3 3 2 2 1 2 12 0 0 1 3 3 3 3 3 3 2 2 1 0 1 0 2 12 0 0 1 3 3 3 3 3 3 2 2 1 0 1 0 2 13 0 1 1 2 3 3 4 3 2 2 2 1 1 1 0 2 13 0 1 1 2 3 3 4 3 2 2 2 1 1 1 0 2 14 0 1 1 2 3 3 4 3 3 3 2 1 1 1 1 2 14 0 1 1 2 3 3 4 3 3 3 2 1 1 1 1 2 15 0 1 1 2 2 3 3 3 2 3 2 1 1 1 1 2 15 0 1 1 2 2 3 3 3 2 3 2 1 1 1 1 2 16 0 1 1 2 3 3 4 3 3 3 2 1 1 1 1 2 16 0 1 1 2 3 3 4 3 3 3 2 1 1 1 1 2 17 0 1 1 1 3 3 2 2 3 2 2 1 0 0 0 2 17 0 1 1 1 3 3 2 2 3 2 2 1 0 0 0 2 18 0 1 1 1 2 3 3 3 2 3 2 0 1 1 1 2 18 0 1 1 1 2 3 3 3 2 3 2 0 1 1 1 2 19 0 0 2 2 3 3 3 3 3 2 2 1 1 1 1 2 19 0 0 2 2 3 3 3 3 3 2 2 1 1 1 1 2 20 0 1 1 2 3 3 4 2 2 2 2 1 1 1 1 2 20 0 1 1 2 3 3 4 2 2 2 2 1 1 1 1 3 21 0 1 1 2 3 3 3 3 2 2 2 1 2 1 1 3 21 0 1 1 2 3 3 3 3 2 2 2 1 2 1 1 3 22 0 2 1 2 3 3 3 2 2 2 2 0 0 0 0 3 22 0 2 1 2 3 3 3 2 2 2 2 0 0 0 0 3 23 0 0 1 2 3 3 3 2 2 2 2 1 0 1 1 3 23 0 0 1 2 3 3 3 2 2 2 2 1 0 1 1 3 24 0 1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 24 0 1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 25 0 1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 25 0 1 1 2 3 3 3 3 2 2 2 1 1 1 1 3 26 0 0 1 2 2 3 3 3 2 2 2 1 1 1 1 3 26 0 0 1 2 2 3 3 3 2 2 2 1 1 1 1 3 27 0 1 1 2 3 3 3 3 3 2 2 1 1 1 1 3 27 0 1 1 2 3 3 3 3 3 2 2 1 1 1 1 3 28 0 1 1 2 3 3 3 3 3 2 2 1 0 1 0 3 28 0 1 1 2 3 3 3 3 3 2 2 1 0 1 0 3 29 0 0 1 2 3 3 3 3 2 2 2 1 2 1 1 3 29 0 0 1 2 3 3 3 3 2 2 2 1 2 1 1 3 30 0 1 1 2 2 3 2 2 2 1 2 0 0 0 0 3 30 0 1 1 2 2 3 2 2 2 1 2 0 0 0 0 4 31 0 0 1 2 3 3 3 3 3 2 2 1 0 1 0 4 31 0 0 1 2 3 3 3 3 3 2 2 1 0 1 0 4 32 0 1 1 2 2 3 3 2 2 3 2 0 1 1 1 4 32 0 1 1 2 2 3 3 2 2 3 2 0 1 1 1 4 33 0 1 1 2 2 3 3 3 3 2 2 0 1 1 1 4 33 0 1 1 2 2 3 3 3 3 2 2 0 1 1 1 4 34 0 1 1 1 2 2 3 3 3 3 2 2 2 2 1 4 34 0 1 1 1 2 2 3 3 3 3 2 2 2 2 1 4 35 0 1 2 2 3 3 3 3 3 3 2 1 0 1 1 4 35 0 1 2 2 3 3 3 3 3 3 2 1 0 1 1 4 36 0 1 1 2 2 3 3 3 3 3 2 1 1 1 1 4 36 0 1 1 2 2 3 3 3 3 3 2 1 1 1 1 4 37 0 1 1 1 2 3 3 3 3 3 2 0 1 1 1 4 37 0 1 1 1 2 3 3 3 3 3 2 0 1 1 1 4 38 0 1 1 2 2 3 3 3 3 3 3 1 1 1 1 4 38 0 1 1 2 2 3 3 3 3 3 3 1 1 1 1 4 39 0 1 1 2 3 3 3 3 3 2 2 1 0 0 0 4 39 0 1 1 2 3 3 3 3 3 2 2 1 0 0 0 4 40 0 1 2 2 3 3 2 2 3 2 2 1 0 0 0 4 40 0 1 2 2 3 3 2 2 3 2 2 1 0 0 0

Example 4 A Study of SCV-07 in the Treatment of Oral Mucositis Induced by a Combination of Cisplatin and Acute Radiation in Hamsters. Introduction

Prior studies with SCV-07 have shown that it is effective in treating oral mucositis in hamster models of the disease induced by both acute and fractionated radiation. In this study, the efficacy of SCV-07 was evaluated in a hamster model of oral mucositis induced by a combination of chemotherapy and radiation, and specifically, by the combination of cis-platin and radiation.

Methods

Forty Golden Syrian hamsters were prospectively randomized into four equally sized groups. Mucositis was induced on the left cheek pouch mucosa of golden Syrian hamsters by a single dose of cis-platin at 5 mg/kg given on day −1, and a single dose of radiation administered on day 0 at a dose of 35Gy. Beginning on day −1 and continuing once daily until day 20, SCV-07 was given by sub-cutaneous injection at doses of 10 μg/kg, 100 μg/kg or 1 mg/kg in a volume of 100 μL. Animals' activity and weight were evaluated daily. Beginning on day 6 and continuing on alternate days for the duration of the study, oral mucositis was evaluated using a standard scoring six point scale. The number of days of ulcerative mucositis was evaluated using a Chi-squared test of scores of ≧3 throughout the study, and the individual daily group scores were assessed with a Rank Sum Test.

Results

Mucositis was favorably and consistently impacted in animals treated with SCV-07 in all test groups. Severe mucositis was reduced from 50% of animals days evaluated in vehicle controls to approximately 30% in SCV-07 treated animals. The most significant impact was seen in the later stages of the disease process, after the peak of mucositis. No significant differences were observed among groups relative to weight changes. No animals died during the course of the experiment.

Conclusions

1. There was no evidence of any toxicity from SCV-07 in this study based on the observations of mortality and weight gain. 2. Animals treated with SCV-07 at 10 μg/kg from day −1 to day 20 showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P<0.001), and a significant reduction in mucositis scores on days 22 (P=0.010), 24 (P=0.022), 26 (P=0.015), and 28 (P<0.001). 3. Animals treated with SCV-07 at 100 μg/kg from day −1 to day 20 showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P<0.001), and a significant reduction in mucositis scores on days 6 (P=0.015), 22 (P=0.003), 24 (P=0.005), 26 (P<0.001), and 28 (P<0.001). 4. Animals treated with SCV-07 at 1 mg/kg from day −1 to day 20 showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P<0.001), and a significant reduction in mucositis scores on days 6 (P=0.015), 20 (P=0.029), 22 (P=0.012), 24 (P=0.001), 26 (P=0.003), and 28 (P<0.001).

1.1 Background

KGF-1 and other FGF family members have been shown to induce epithelial thickening of the oral and esophageal mucosal surfaces in BDF-1. SCV-07 and the derived SCV-07 peptide are believed to have mechanisms that that may overlap with KGF-1, and have been shown to be protective in other models of mucosal injury.

Oral ulcerative mucositis is a common, painful, dose-limiting toxicity of drug and radiation therapy for cancer. The disorder is characterized by breakdown of the oral mucosa that results in the formation of ulcerative lesions. In granulocytopenic patients, the ulcerations that accompany mucositis are frequent portals of entry for indigenous oral bacteria often leading to sepsis or bacteremia. Mucositis occurs to some degree in more than one third of patients receiving anti-neoplastic drug therapy. The frequency and severity are significantly greater among patients who are treated with induction therapy for leukemia or with many of the conditioning regimens for bone marrow transplant. Among these individuals, moderate to severe mucositis is not unusual in more than three-quarters of patients. Moderate to severe mucositis occurs in virtually all patients who receive radiation therapy for tumors of the head and neck and typically begins with cumulative exposures of 15 Gy and then worsens as total doses of 60 Gy or more are reached.

Clinically mucositis progresses through three stages:

1. Painful erythema which can generally be managed by topical anesthetics or non-narcotic analgesics. 2. Painful ulceration often with pseudomembrane formation. In the case of concomitant myelosuppression, bacteremias or sepsis of oral origin are not uncommon. Pain is often of such intensity as to require narcotic analgesia, frequently parenterally. 3. Spontaneous healing, occurring about 2-3 weeks after cessation of anti-neoplastic therapy.

Currently, the only approved biologic or drug for mucositis prevention and/or treatment is Kepivance (palifermin). Kepivance use is limited to mucositis in patients receiving stem cell transplant for hematologic malignancies. Consequently, standard therapy for mucositis consists of palliative rinses, such as saline, bicarbonate solutions, mouthwashes, topical analgesics such as lidocaine and/or systemic administration of narcotics.

The complexity of mucositis as a biological process has only been recently appreciated. It has been suggested that the condition represents a sequential interaction of oral mucosal cells and tissues reactive oxygen species, pro-inflammatory cytokines, mediators of apoptosis, a range of signaling pathways, and local factors such as saliva and the oral micro biota. While epithelial degeneration and breakdown ultimately result in mucosal ulceration, it appears that the early changes associated with radiation-induced mucosal toxicity occur within the endothelium, and connective tissue of the submucosa. It appears that the overall mechanism for mucositis development is similar for both radiation and chemotherapy.

1.2 Chemo-Radiation Model of Oral Mucositis with Cis-Platin.

The chemo-radiation model of oral mucositis in hamsters, developed by the Principal Investigator, has proven to be an accurate, efficient and cost-effective technique to provide a preliminary evaluation of anti-mucositis compounds. In this model, hamsters received a single dose of cis-platin at 5 mg/kg on day −1, followed by a single dose of radiation of 35 Gy to the left cheek pouch on day 0, rather than the single dose of 40 Gy on day 0 that is used in acute radiation studies. The course of mucositis in this model is very similar to the acute radiation model and results in peak mucositis scores approximately 16-18 days following radiation.

2. Study Objective and Summary 2.1 Study Objective

The objective of this study was to evaluate the effect of SCV-07, administered by sub-cutaneous injection, on the frequency, severity and duration of oral mucositis induced by a fractionated radiation protocol.

2.2 Study Summary

Hamsters received a single dose of cis-platin at 5 mg/kg on day −1 by intraperitoneal injection, followed by a single dose of radiation of 35 Gy to the left cheek pouch on day 0. This was accomplished by anesthetizing the animals and everting the left buccal pouch, while protecting the rest of the animal's bodies with a lead shield. Test materials were given by sub-cutaneous injection once daily as detailed in Table 13. Mucositis was evaluated clinically starting on day 6, and continuing on alternate days until day 28. Test articles were given as from day −1 to day 20.

3. Evaluation 3.1 Mucositis Evaluation

The grade of mucositis was scored, beginning day 6, and for every second day thereafter, through and including day 28. The effect on mucositis of each drug treatment compared to placebo was assessed according to the following parameters:

3.1.1 The Difference in the Number of Days Hamsters in Each Group have Ulcerative (Score ≧3) Mucositis.

On each evaluation day, the number of animals with a blinded mucositis score of ≧3 in each drug treatment group was compared to the control group. Differences were compared on a cumulative basis and statistical significance was determined by chi-square analysis. Efficacy, in this analysis, is defined by a significant reduction in the number of days that a group of animals had ulcerations (scores ≧3) when compared to the control group.

3.1.2 Rank Sum Differences in Daily Mucositis Scores.

For each evaluation day the scores of the control group were compared to those of the treated groups using non-parametric rank sum analysis. Treatment success was considered as a statistically significant lowering of scores in the treated group on 2 or more days from day 6 to day 28.

3.2 Weights and Survival

All animals were weighed daily and their survival recorded, in order to assess possible differences in animal weight among treatment groups as an indication for mucositis severity and/or possible toxicity resulting from the treatments.

4. Study Design

Forty (40) male Syrian Golden Hamsters were given an intraperitoneal injection of 5 mg/kg Cisplatin on day −1. On day 0 all animals were given an acute radiation dose of 35 Gy directed to their left buccal cheek pouch. This was accomplished by anesthetizing the animals and everting the left buccal pouch, while protecting the rest of the animals with a lead shield. Test materials were given by subcutaneous injection once daily as detailed in Table 13. Mucositis was evaluated clinically starting on Day 6, and continued on alternate days until day 28.

TABLE 13 SCI-04. Study Design Number of Treatment Volume Group Animals Cisplatin Radiation Treatment Schedule* (mL) 1 10 males 5 mg/kg, 35 Gy, day 0 Vehicle (PBS), sc, qd Day −1 to 20 Adjust per day −1 body weight 2 10 males 5 mg/kg, 35 Gy, day 0 SCV-07, sc, qd Day −1 to 20 Adjust per day −1 10 μg/kg, body weight 3 10 males 5 mg/kg, 35 Gy, day 0 SCV-07, sc, qd Day −1 to 20 Adjust per day −1 100 μg/kg, body weight 4 10 males 5 mg/kg, 35 Gy, day 0 SCV-07, sc, qd Day −1 to 20 Adjust per day −1 1.0 mg/kg body weight *The dose on day 0 will be performed 30 minutes prior to radiation

5. Material and Methods 5.1 Location of Study Performance

The study was performed at Biomodels AAALAC-accredited facility in Watertown, Mass. IACUC approval number 07-0620-01 for this study was obtained from Biomodels IACUC.

5.2 Animals

Male LVG Syrian Golden Hamsters (Charles River Laboratories), aged 5 to 6 weeks, with average body weight of 90 g at study commencement, were used. Animals were individually numbered using an ear punch and housed in small groups of approximately 10 animals per cage. Animals were acclimatized for 5 days prior to study commencement and during this period, the animals were observed daily in order to reject animals that present in poor condition.

5.3 Housing

The study was performed in animal rooms provided with filtered air at a temperature of 70° F.+/−5° F. and 50%+/−20% relative humidity. Animal rooms were set to maintain a minimum of 12 to 15 air changes per hour. The room was on an automatic timer for a light/dark cycle of 12 hours on and 12 hours off with no twilight. Bed-O-Cobs® bedding was used. Bedding was changed a minimum of once per week. Cages, tops, bottles, etc. were washed with a commercial detergent and allowed to air dry. A commercial disinfectant was used to disinfect surfaces and materials introduced into the hood. Floors were swept daily and mopped a minimum of twice weekly with a commercial detergent. Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution. A cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity was recorded during the study, and the records retained.

5.4 Diet

Animals were fed with a Purina Labdiet® 5061 rodent diet and water was provided ad libitum.

5.5 Animal Randomization and Allocations.

Animals were randomly and prospectively divided into four (4) treatment groups prior to irradiation. Each animal was identified by an ear punch corresponding to an individual number. For more consistent identification, ear punch numbering was used rather than tagging, since tags may become dislodged during the course of the study. A cage card was used to identify each cage or label marked with the study number (SCI-04), treatment group number and animal numbers.

5.6 Sub-Cutaneous Dosing and Drug Application

The test compound, human SCV-07 peptide was provided as a powder and dissolved in sterile PBS immediately prior to administration. Drug was given in a volume of 0.1, using a tuberculin syringe with a 27G needle. Injections were given subcutaneously to the back or abdomen.

5.7 Mucositis Induction

In this study, mucositis was induced with a combination of Cis-platin and radiation. Cis-platin was given as a single injection (IP) of 5 mg/kg on day −1. Radiation was given as a single focal dose of 35 Gy on day 0. Radiation was generated with a Philips 160 kVp (kilovolt potential) (18.75-ma) X-ray source at a focal distance of 30 cm, with a 3.0 mm hardened Al filtration system. Irradiation was targeted to the left buccal pouch mucosa at a rate of 3.32 Gy/minute. Calibration of this source with a Victoreen model 530 dosimeter indicated that the dose rate was 28.57 nC/min. Using this calibration, the energy received by each animal at each radiation dose was approximately 688.6 nC (nanoCoulombs). Prior to irradiation, animals were anesthetized with an intraperitoneal injection of ketamine (160 mg/kg) and xylazine (8 mg/kg). The left buccal pouch was everted, fixed and isolated using a lead shield.

5.8 Mucositis Scoring

The mucositis score, weight change and survival were measured throughout the study as described above. For the evaluation of mucositis, the animals were anesthetized with an inhalation anesthetic, and the left pouch everted. Mucositis was scored visually by comparison to a validated photographic scale, ranging from 0 for normal, to 5 for severe ulceration (clinical scoring). In descriptive terms, this scale is defined as follows:

TABLE 14 SCI-04: Mucositis Scoring. Score: Description: 0 Pouch completely healthy. No erythema or vasodilation. 1 Light to severe erythema and vasodilation. No erosion of mucosa. 2 Severe erythema and vasodilation. Erosion of superficial aspects of mucosa leaving denuded areas, but no frank ulceration. Decreased stippling of mucosa. 3 Formation of off-white ulcers in one or more places. Ulcers may have a yellow/gray due to pseudomembrane. Cumulative size of ulcers should equal about ¼ of the pouch. Severe erythema and vasodilation. 4 Cumulative seize of ulcers should equal about ½ of the pouch. Loss of pliability. Severe erythema and vasodilation. 5 Virtually all of pouch is ulcerated. Loss of pliability (pouch can only partially be extracted from mouth).

A score of 1-2 is considered to represent a mild stage of the disease, whereas a score of 3-5 is considered to indicate moderate to severe mucositis. Following visual scoring, a photograph was taken of each animal's mucosa using a standardized technique. At the conclusion of the experiment, all films were developed and the photographs randomly numbered. At least two independent trained observers graded the photographs in blinded fashion using the above-described scale (blinded scoring).

6. Results and discussion 6.1 Survival

No deaths were seen in this study

6.2 Weight Change

There were no significant differences in weight changes between study groups. The mean daily percent weight change data was evaluated. The saline treated control hamsters gained an average of 46.5% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 10 μg/kg on days −1 to 20 gained an average of 51.3% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 100 μg/kg on days −1 to 20 gained an average of 46.7% of their starting weight during the study. Hamsters in the group receiving SCV-07 at 1 mg/kg from day −1 to day 20 gained an average of 48.8% of their starting weight during the study. The significance of these differences was evaluated by calculating the area-under-the-curve (AUC) for the weight gain of each animal, and then comparing the different treatment groups using a One-Way ANOVA test. The results of this analysis indicated that there were no significant differences between the different treatment groups (P=0.663).

6.3 Mucositis (Tables 15 & 16)

The kinetics and severity of mucositis development among control animals was consistent with that which was expected.

Mean daily mucositis scores for each group were evaluated. In the saline treated control group, the mean peak mucositis score was 3.1, which occurred on day 18. The group receiving SCV-07 at 10 μg/kg from day −1 to day 20 had a peak mean mucositis score of 2.9, which occurred on day 16. The group receiving SCV-07 at 100 μg/kg from day −1 to day 20 had a peak mean mucositis score of 2.8, which occurred on days 14 and 16. The group receiving SCV-07 at 1 mg/kg from day −1 to day 20 had a peak mean mucositis score of 3.2, which occurred on day 16. The significance of the differences observed between the different treatment groups was evaluated by calculating the number of days with a score of 3 or higher for each group and comparing these numbers using a chi-squared (□²) test. The results of this analysis are shown in Table 15. The hamsters in the saline treated control group had a score of 3 or higher on 50% of the animal days evaluated. In the group receiving SCV-07 at 10 μg/kg from day −1 to day 20, a mucositis score of 3 or higher was observed on 34.2% of the animals days evaluated, which was not statistically significantly different from controls (P<0.001). In the group receiving SCV-07 at 100 μg/kg on days −1 to 20, a mucositis score of 3 or higher was observed on 29.2% of the animals days evaluated, which was statistically significantly different from controls (P<0.001). In the group receiving SCV-07 at 100 μg/kg on days −1 to 20, a mucositis score of 3 or higher was observed on 30.8% of the animals days evaluated, which was not statistically significantly different from controls (P<0.001). A further analysis of the mucositis scores was performed using the Mann-Whitney rank sum analysis to compare the scores for each group on each day. The results of this analysis are shown in Table 16. In this analysis, 2 days of significant reduction in the mucositis score are generally required before it is regarded as meaningful. The group treated with SCV-07 at 10 μg/kg on days −1 to 20, had significantly less mucositis than the saline controls on days 22 (P=0.010), 24 (P=0.022), 26 (P=0.015), and 28 (P<0.001). The group treated with SCV-07 at 100 μg/kg on days −1 to 20, had significantly less mucositis than the saline controls on days 6 (P=0.015), 22 (P=0.003), 24 (P=0.005), 26 (P<0.001), and 28 (P<0.001). The group treated with SCV-07 at 1 mg/kg on days −1 to 20, had significantly less mucositis than the saline controls on days 6 (P=0.015), 20 (P=0.029), 22 (P=0.012), 24 (P=0.001), 26 (P=0.003), and 28 (P<0.001).

TABLE 15 SCI-04. Chi-square analysis of the total number of days the animals in each group spent with a score of three or more. This statistic is a measure of severity of ulceration, a clinically important outcome. Days Days Total % Days Chi Sq Group >=3 <3 Days >=3 v control P Value Vehicle, qd, sc 120 120 240 50.0 — — Days −1 to 20 SCV-07 82 158 240 34.2 11.702 <0.001 10 ug/kg, qd, sc Days −1 to 20 SCV-07 70 170 240 29.2 20.9160 <0.001 100 ug/kg, qd, sc Days −1 to 20 SCV-07 74 166 240 30.8 17.5190 <0.001 1 mg/kg, qd, sc Days −1 to 20

TABLE 16 SCI-04. The significance of group differences observed in daily mucositis scores was determined using the Mann-Whitney rank sum test. This nonparametric statistic is appropriate for the visual mucositis scoring scale. The p values for each calculation are shown. Significant reductions in mucositis scores relative to controls are shown underlined. Day Group Comparison 6 8 10 12 14 16 18 20 22 24 26 28 Control vs 0.296 0.422 0.393 0.560 0.271 0.488 0.179 0.100 0.010 0.022 0.015 <0.001 SCV-7 10 ug/kg, qd, sc Days −1 to 20 Control vs 0.015 0.422 0.510 0.957 0.506 0.248 0.100 0.455 0.003 0.005 <0.001  <0.001 SCV-7 100 ug/kg, qd, sc Days −1 to 20 Control vs 0.015 0.282 0.146 0.924 0.506 0.634 0.112 0.029 0.012 0.001 0.003 <0.001 SCV-7 1 mg/kg, qd, sc Days −1 to 20

7. Conclusions

1. There was no evidence of any toxicity from SCV-07 in this study based on the observations of mortality and weight gain. 2. Animals treated with SCV-07 at 10 μg/kg from day −1 to day 20 showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P<0.001), and a significant reduction in mucositis scores on days 22 (P=0.010), 24 (P=0.022), 26 (P=0.015), and 28 (P<0.001). 3. Animals treated with SCV-07 at 100 μg/kg from day −1 to day 20 showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P<0.001), and a significant reduction in mucositis scores on days 6 (P=0.015), 22 (P=0.003), 24 (P=0.005), 26 (P<0.001), and 28 (P<0.001). 4. Animals treated with SCV-07 at 1 mg/kg from day −1 to day 20 showed a statistically significant reduction in the number of days with a mucositis score of 3 or higher (P<0.001), and a significant reduction in mucositis scores on days 6 (P=0.015), 20 (P=0.029), 22 (P=0.012), 24 (P=0.001), 26 (P=0.003), and 28 (P<0.001).

9. Appendices

APPENDIX 7 Animal Weights DAY Group Animal −1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 1 105 104 100 103 106 108 111 111 113 113 115 116 118 121 119 121 1 2 88 86 83 89 92 94 96 97 101 103 106 105 104 103 104 108 1 3 90 92 88 91 95 99 101 103 104 92 105 109 109 108 107 109 1 4 87 86 83 87 90 93 96 98 100 102 105 106 106 109 109 110 1 5 106 101 99 105 107 111 113 113 117 118 120 122 120 121 120 124 1 6 82 83 79 82 84 86 87 89 90 97 96 97 97 98 96 95 1 7 86 85 84 85 88 90 93 94 95 97 98 99 100 101 103 104 1 8 82 81 78 83 86 89 92 93 98 105 105 103 103 107 106 107 1 9 90 89 91 93 95 101 101 103 104 106 107 111 112 113 114 118 1 10 108 109 104 105 110 112 117 118 120 122 123 126 128 129 124 126 2 11 77 77 74 78 82 83 86 88 90 92 94 96 98 98 98 99 2 12 101 102 101 104 111 113 112 114 117 121 122 125 125 128 128 130 2 13 85 84 83 89 93 95 98 101 103 106 106 112 111 111 112 115 2 14 82 80 80 86 88 90 93 96 98 101 102 105 105 107 108 109 2 15 76 77 76 77 79 82 83 84 86 88 91 90 92 91 90 91 2 16 94 94 91 94 98 101 103 106 108 109 108 109 109 109 108 111 2 17 74 72 70 77 79 76 81 79 80 83 83 85 87 88 88 90 2 18 80 78 75 78 81 85 85 89 89 92 92 94 95 94 95 96 2 19 91 90 87 90 91 93 94 94 97 98 98 100 100 97 98 99 2 20 92 92 90 95 96 99 100 102 105 106 108 110 111 113 114 114 3 21 87 84 87 90 94 98 99 98 100 103 106 111 109 111 111 110 3 22 81 80 75 77 81 84 86 88 87 90 91 95 95 98 96 97 3 23 85 86 83 86 88 91 93 96 95 98 96 101 100 102 102 103 3 24 104 102 99 102 106 109 113 112 116 118 118 123 123 126 127 130 3 25 78 76 73 76 82 83 84 84 87 90 89 95 94 92 92 95 3 26 92 91 88 90 91 92 95 95 96 98 98 100 100 101 100 100 3 27 103 103 102 100 108 110 115 118 119 120 120 124 124 127 125 128 3 28 96 96 91 90 101 114 105 106 107 108 111 113 114 117 116 116 3 29 83 82 84 87 91 91 94 94 97 98 97 103 101 102 102 101 3 30 77 78 74 76 78 79 81 83 86 88 88 92 94 93 94 94 4 31 97 96 94 98 103 106 108 110 111 112 112 113 111 113 113 114 4 32 96 96 93 95 99 103 104 106 109 110 111 113 113 113 114 113 4 33 93 91 89 90 94 100 101 103 107 110 110 114 115 114 113 113 4 34 87 85 85 89 92 96 98 103 104 120 107 109 112 113 113 115 4 35 100 96 97 101 106 108 111 115 118 105 121 124 122 126 124 124 4 36 88 88 85 87 93 96 99 103 105 108 109 113 114 118 120 121 4 37 104 102 104 108 114 115 120 119 123 125 127 128 127 127 122 125 4 38 97 96 93 93 98 103 105 107 110 113 113 116 116 116 115 114 4 39 88 88 83 85 93 93 95 95 98 100 102 104 102 104 104 103 4 40 99 97 99 101 107 107 112 113 115 117 118 120 123 125 125 127 DAY Group Animal 15 16 17 18 19 20 21 22 23 24 25 26 27 28 1 1 119 121 124 125 126 127 130 133 133 137 137 138 139 140 1 2 112 110 117 116 120 121 124 125 123 131 134 136 135 139 1 3 112 117 116 117 118 120 126 127 129 131 132 135 134 138 1 4 110 109 113 115 115 115 121 120 121 126 126 129 129 133 1 5 123 125 129 129 129 130 133 136 135 141 141 143 144 144 1 6 95 96 97 99 99 100 102 107 104 106 107 108 109 111 1 7 103 104 107 107 108 108 111 113 113 115 118 120 120 122 1 8 108 109 113 115 115 115 119 122 123 125 127 128 129 132 1 9 118 117 122 123 123 126 128 130 131 134 135 136 138 138 1 10 126 128 131 134 134 135 137 139 141 144 147 148 148 151 2 11 99 100 103 103 105 104 108 110 110 114 115 117 117 119 2 12 132 132 136 137 138 137 141 144 143 148 150 151 152 156 2 13 117 119 123 125 125 127 130 133 133 139 139 140 145 146 2 14 109 109 112 113 113 115 117 120 121 123 124 126 127 130 2 15 93 92 96 98 100 100 103 104 104 105 108 109 110 113 2 16 112 112 117 117 118 118 121 122 123 126 126 129 128 132 2 17 90 90 96 97 99 101 105 104 106 109 113 114 114 117 2 18 97 96 103 102 101 109 105 108 108 111 112 116 114 116 2 19 98 99 100 104 103 104 106 107 108 111 113 115 113 115 2 20 115 117 121 122 123 123 127 131 129 134 136 137 138 143 3 21 109 110 112 114 115 118 117 123 123 128 127 130 130 133 3 22 97 98 100 102 103 104 107 109 108 113 112 115 112 117 3 23 103 103 106 107 110 110 113 114 113 117 117 123 119 123 3 24 131 132 134 134 140 139 142 146 146 146 148 149 149 154 3 25 95 97 99 101 101 105 106 108 106 113 115 118 116 120 3 26 110 101 103 104 105 106 106 109 109 112 112 113 113 116 3 27 128 128 129 132 134 136 138 139 138 141 143 146 146 149 3 28 116 117 119 120 123 123 126 128 128 131 133 132 133 137 3 29 103 105 106 108 112 112 117 118 117 123 123 125 126 130 3 30 95 97 100 101 102 102 104 107 107 111 114 116 115 118 4 31 112 113 114 116 117 120 121 123 124 130 130 133 133 136 4 32 123 116 118 119 119 122 123 125 124 129 130 132 133 136 4 33 112 114 117 119 122 122 126 129 128 133 137 138 138 141 4 34 116 116 120 120 123 122 125 126 127 129 131 131 131 133 4 35 114 123 127 130 130 132 137 141 139 142 147 147 145 150 4 36 118 119 120 123 123 125 128 130 129 134 136 139 137 142 4 37 125 128 131 132 134 135 139 143 143 145 148 150 150 153 4 38 115 115 119 119 121 122 123 125 126 128 130 133 132 135 4 39 105 107 110 112 114 114 115 117 116 119 123 126 125 130 4 40 125 128 131 134 135 133 138 142 144 148 149 152 151 155

APPENDIX 8 Mucositis Scores Ani- DAY Group mal 6 8 10 12 14 16 18 20 22 24 26 28 1 1 2 0 1 2 3 3 3 3 3 3 3 3 1 1 1 0 1 2 3 3 3 3 3 3 3 3 1 2 1 1 2 3 3 3 3 3 3 2 2 2 1 2 0 0 2 3 3 3 4 3 3 2 2 2 1 3 2 1 2 3 4 4 3 3 3 3 3 3 1 3 1 1 2 3 3 4 3 3 3 3 3 3 1 4 0 0 1 2 2 3 3 3 3 3 3 3 1 4 0 0 1 2 2 3 3 3 3 3 3 3 1 5 2 1 2 3 3 3 3 3 3 3 3 3 1 5 1 1 2 2 3 3 3 3 3 3 3 3 1 6 1 0 1 2 3 3 3 3 3 2 2 2 1 6 0 0 1 2 3 3 3 3 3 2 2 2 1 7 0 0 1 1 2 2 2 2 2 2 1 1 1 7 0 0 0 1 2 2 2 2 2 2 1 1 1 8 1 0 1 2 3 3 3 3 3 2 2 2 1 8 0 0 1 2 3 3 4 3 3 2 2 2 1 9 0 0 1 3 3 3 3 3 3 2 2 1 1 9 0 0 1 3 3 3 3 3 2 2 2 1 1 10 0 0 1 2 4 3 4 3 3 3 2 2 1 10 0 0 0 2 4 4 4 3 3 3 2 2 2 11 0 0 1 2 2 3 2 3 2 2 1 0 2 11 0 0 1 2 2 3 2 3 2 1 1 0 2 12 1 0 2 2 2 2 2 2 1 1 1 0 2 12 0 0 1 2 2 2 2 2 1 1 1 0 2 13 1 0 1 3 3 3 3 2 2 2 1 0 2 13 0 0 1 3 3 3 3 1 2 2 1 0 2 14 1 0 1 2 2 3 3 3 3 3 3 2 2 14 0 0 1 2 2 3 3 3 3 3 3 2 2 15 1 0 1 2 3 3 3 3 3 2 2 1 2 15 0 0 1 2 3 3 3 3 3 2 1 1 2 16 1 1 2 3 3 2 2 2 1 1 1 0 2 16 0 0 2 3 3 2 2 2 1 1 1 1 2 17 1 0 2 3 3 4 3 3 2 2 2 1 2 17 0 0 2 3 3 4 3 3 2 2 2 1 2 18 0 0 2 2 3 3 3 2 2 3 1 1 2 18 0 0 1 2 3 3 3 2 2 3 1 1 2 19 0 0 1 3 3 3 4 3 3 2 3 3 2 19 0 0 1 3 3 3 4 3 3 2 3 3 2 20 0 1 2 2 3 3 3 3 3 2 2 2 2 20 0 0 2 2 3 3 3 3 3 2 2 1 

1-16. (canceled)
 17. A method for reducing the amount of mucosal deterioration, injury, or damage to at least one of oral and esophageal mucosal tissues, the mucosal deterioration, injury, or damage resulting from radiation treatment in a subject undergoing radiation therapy for head and neck cancer, the method comprising: administering to said subject an effective amount of a compound of the formula

or a pharmaceutically acceptable salt thereof, wherein: n is 1 or 2; R is hydrogen or a C2-10 acyl or C1-6 alkyl; and X is L-tryptophan or D-tryptophan.
 18. The method of claim 17, wherein said compound is not γ-D-glutamyl-L-tryptophan (SCV-07) or a pharmaceutically acceptable salt thereof.
 19. The method of claim 17, wherein the oral and/or esophageal mucosal deterioration, injury, or damage comprises ulcerative lesions.
 20. The method of claim 17, wherein said radiation is administered at a cumulative dose of at least about 15 Gy.
 21. The method of claim 17, wherein said radiation is administered at a cumulative dose of at least about 40 Gy.
 22. The method of claim 17, wherein said radiation is administered at a cumulative dose of at least about 60 Gy.
 23. The method of claim 17, wherein a chemotherapy is administered to the subject.
 24. The method of claim 23, wherein the chemotherapy is cis-platin.
 25. The method of claim 24, wherein said cis-platin is administered at a dosage within a range of 0.1-50 mg/kg.
 26. The method of claim 24, wherein said cis-platin is administered at a dosage of about 5 mg/kg.
 27. The method of claim 17 wherein said compound or a pharmaceutically acceptable salt thereof is administered before radiation exposure.
 28. The method of claim 17 wherein said compound or a pharmaceutically acceptable salt thereof is administered during radiation exposure.
 29. The method of claim 17 wherein said compound or a pharmaceutically acceptable salt thereof is administered after radiation exposure.
 30. The method of claim 17 wherein said compound or a pharmaceutically acceptable salt thereof is administered before administration of 7-8 Gy of radiation. 